Subtilase Variants

ABSTRACT

The present invention relates to enzymes produced by mutating the genes for a number of subtilases and expressing the mutated genes in suitable hosts are presented. The enzymes exhibit improved stability and/or improved wash performance in any detergent in comparison to their wild type parent enzymes. The enzymes are well-suited for use in any detergent and for some in especially liquid or solid shaped detergent compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/618,934filed Jul. 14, 2003, which is a continuation of application Ser. No.09/587,747 filed Jun. 5, 2000, now U.S. Pat. No. 6,682,924, which is acontinuation of application Ser. No. 09/120,577 filed Jul. 22, 1998, nowU.S. Pat. No. 6,190,900, which is a continuation of application Ser. No.08/642,987 filed May 6, 1996, now U.S. Pat. No. 5,837,517, which claimspriority under 35 U.S.C. 119 of Danish application nos. 0519/95 and0421/96 filed May 5, 1995 and Apr. 12, 1996, respectively, and Europeanapplication no. 95201161 filed May 5, 1995, the contents of which arefully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a paper copy and computer readable form of aSequence Listing, which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel mutant enzymes or enzyme variants usefulin formulating detergent compositions and exhibiting improved storagestability while retaining or improving their wash performance; cleaningand detergent compositions containing said enzymes; mutated genes codingfor the expression of said enzymes when inserted into a suitable hostcell or organism; and such host cells transformed therewith and capableof expressing said enzyme variants.

2. Description of Related Art

In the detergent industry enzymes have for more than 30 years beenimplemented in washing formulations. Enzymes used in such formulationscomprise proteases, lipases, amylases, cellulases, as well as otherenzymes, or mixtures thereof. Commercially most important are proteases.

Although proteases have been used in the detergent industry for morethan 30 years, much remains unknown as to details of how these enzymesinteract with substrates and/other substances present in e.g. detergentcompositions. Some factors related to specific residues and influencingcertain properties, such as oxidative and thermal stability in generalhave been elucidated, but much remains to be found out. Also, it isstill not exactly known which physical or chemical characteristics areresponsible for a good washing performance or stability of a protease ina specific detergent composition.

The currently used proteases have for the most part been found byisolating proteases from nature and testing them in detergentformulations.

An increasing number of commercially used protease are proteinengineered variants of the corresponding naturally occurring wild typeprotease, e.g. DURAZYM® (Novo Nordisk A/S), RELASE® (Novo Nordisk A/S),MAXAPEM® (Gist-Brocades N.V.), PURAFECT® (Genencor International, Inc.).

Therefore, an object of the present invention is to provide improvedprotein engineered protease variants, especially for use in thedetergent industry.

Proteases

Enzymes cleaving the amide linkages in protein substrates are classifiedas proteases, or (interchangeably) peptidases (see Walsh, 1979,Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco,Chapter 3). Bacteria of the Bacillus species secrete two extracellularspecies of protease, a neutral, or metalloprotease, and an alkalineprotease which is functionally a serine endopeptidase and usuallyreferred to as subtilisin. Secretion of these proteases has been linkedto the bacterial growth cycle, with greatest expression of proteaseduring the stationary phase, when sporulation also occurs. Joliffe etal. (1980) J. Bacteriol 141 1199-1208, have suggested that Bacillusproteases function in cell wall turnover.

Subtilases

A serine protease is an enzyme which catalyzes the hydrolysis of peptidebonds, and in which there is an essential serine residue at the activesite (White, Handler and Smith, 1973 “Principles of Biochemistry,” FifthEdition, McGraw-Hill Book Company, NY, pp. 271-272).

The bacterial serine proteases have molecular weights in the 20,000 to45,000 Daltons range. They are inhibited by diisopropylfluorophosphate.They hydrolyze simple terminal esters and are similar in activity toeukaryotic chymotrypsin, also a serine protease. A more narrow term,alkaline protease, covering a sub-group, reflects the high pH optimum ofsome of the serine proteases, from pH 9.0 to 11.0 (for review, seePriest (1977) Bacteriological Rev. 41 711-753).

A sub-group of the serine proteases tentatively designated subtilaseshas been proposed by Siezen et al., Protein Engng. 4 (1991) 719-737.They are defined by homology analysis of more than 40 amino acidsequences of serine proteases previously referred to as subtilisin-likeproteases. A subtilisin was previously defined as a serine proteaseproduced by Gram-positive bacteria or fungi, and according to Siezen etal., now is a subgroup of the subtilases. A wide variety of subtilisinshave been identified, and the amino acid sequence of a number ofsubtilisins has been determined. These include more than six subtilisinsfrom Bacillus strains, namely, subtilisin 168, subtilisin BPN′,subtilisin Carlsberg, subtilisin Y, subtilisin amylosacchariticus, andmesentericopeptidase (Kurihara et al. (1972) J. Biol. Chem. 2475629-5631; Wells et al. (1983) Nucleic Acids Res. 11 7911-7925; Stahland Ferrari (1984) J. Bacteriol. 159 811-819, Jacobs et al. (1985) Nucl.Acids Res. 13 8913-8926; Nedkov et al. (1985) Biol. Chem. Hoppe-Seyler366 421-430, Svendsen et al. (1986) FEBS Lett. 196 228232), onesubtilisin from an actinomycetales, thermitase from Thermoactinomycesvulgaris (Meloun et al., (1985) FEBS Lett. 198 195-200), and one fungalsubtilisin, proteinase K from Tritirachium album (Jany and Mayer (1985)Biol. Chem. Hoppe-Seyler 366 584-492). for further reference Table Ifrom Siezen et al., has been reproduced below.

Subtilisins are well-characterized physically and chemically. Inaddition to knowledge of the primary structure (amino acid sequence) ofthese enzymes, over 50 high resolution X-ray structures of subtilisinshave been determined which delineate the binding of substrate,transition state, products, at least three different proteaseinhibitors, and define the structural consequences for natural variation(Kraut (1977) Ann. Rev. Biochem. 46 331-358).

In the context of this application substrate should be interpreted inits broadest form as comprising a compound containing at least onepeptide bond susceptible to hydrolysis by a subtilisin protease.

Also the expression “product” should in the context of this invention beinterpreted to include the products of a hydrolysis reaction involving asubtilisin protease. A product may be the substrate in a subsequenthydrolysis reaction.

One subgroup of the subtilases, I-S1, comprises the “classical”subtilisins, such as subtilisin 168, subtilisin BPN′, subtilisinCarlsberg (ALCALASE®, Novo Nordisk A/S), and subtilisin DY.

A further subgroup of the subtilases I-S2 is recognised by Siezen etal., (supra). Sub-group I-S2 proteases are described as highly alkalinesubtilisins and comprise enzymes such as subtilisin PB92 (MAXACAL®,Gist-Brocades NV), subtilisin 309 (SAVINASE®, Novo Nordisk A/S),subtilisin 147 (ESPERASE®, Novo Nordisk A/S), and alkaline elastase YaB.

In the context of this invention, a subtilase variant or mutatedsubtilase means a subtilase that has been produced by an organism whichis expressing a mutant gene derived from a parent microorganism whichpossessed an original or parent gene and which produced a correspondingparent enzyme, the parent gene having been mutated in order to producethe mutant gene from which said mutated subtilisin protease is producedwhen expressed in a suitable host.

Random and site-directed mutations of the subtilase gene have botharisen from knowledge of the physical and chemical properties of theenzyme and contributed information relating to subtilase's catalyticactivity, substrate specificity, tertiary structure, etc. (Wells et al.(1987) Proc. Natl. Acad. Sci. U.S.A. 84; 1219-1223; Wells et al. (1986)Phil. Trans. R. Soc. Lond. A. 317 415-423; Hwang and Warshel (1987)Biochem. 26 26692673; Rao et al., (1987) Nature 328 551-554.

More recent publications covering this area are Carter et al., (1989)Proteins 6 240-248 relating to design of variants that cleave a specifictarget sequence in a substrate (positions 24 and 64); Graycar et al.,(1992) Annals of the New York Academy of Sciences 672 71-79 discussing anumber of previously published results; and Takagi (1993) Int. J.Biochem. 25 307-312 also reviewing previous results.

Especially site-directed mutagenesis of the subtilisin genes hasattracted much attention, and various mutations are described in thefollowing patent applications and patents:

EP 130 756 (Genentech) (corresponding to US Reissue Pat. No. 34,606(Genencor)) relating to site specific or randomly generated mutations in“carbonyl hydrolases” and subsequent screening of the mutated enzymesfor various properties, such as k_(cat)/K_(m) ratio, pH-activityprofile, and oxidation stability. This publication reveals thatsite-specific mutation is feasible, and that mutation of subtilisin BPN′in certain specified positions, i.e. ⁻¹Tyr, ³²Asp, ¹⁵⁵Asn, ¹⁰⁴Tyr,²²²Met, ¹⁶⁶Gly, ⁶⁴His, ¹⁶⁹Gly, ¹⁸⁹Phe, ³³Ser, ²²¹Ser, ²⁷Tyr, ¹⁵⁶Glu or¹⁵²Ala, provide for enzymes exhibiting altered properties. Since thesepositions all except position −1 were known to be involved in thefunctioning of the enzyme prior to the filing of the application, andtherefore evident to select, this application does not contribute muchto solving the problem of deciding where to introduce mutations in orderto obtain enzymes with desired properties.

EP 214 435 (Henkel) relates to cloning and expression of subtilisinCarlsberg and two mutants thereof. In this application no reason formutation of ¹⁵⁸Asp to ¹⁵⁸Ser and ¹⁶¹Ser to ¹⁶¹Asp is provided.

International patent publication No. WO 87/04461 (Amgen) proposes toreduce the number of Asn-Gly sequences present in the parent enzyme inorder to obtain mutated enzymes exhibiting improved pH and heatstabilities, in the application emphasis is put on removing, mutating,or modifying the ¹⁰⁹Asn and the ²¹⁸Asn residues in subtilisin BPN′. Noexamples are provided for any deletions or for modifying theGly-residues.

International patent publication No. WO 87/05050 (Genex) disclosesrandom mutation and subsequent screening of a large number of mutants ofsubtilisin BPN′ for improved properties. In the application mutationsare described in positions ²¹⁸Asn, ¹³¹Gly, ²⁵⁴Thr, ¹⁶⁶Gly, ¹¹⁶Ala,¹⁸⁸Ser, ¹²⁶Leu, and ⁵³Ser.

EP 251 446 (Genencor) describes how homology considerations at bothprimary and tertiary structural levels may be applied to identifyequivalent amino acid residues whether conserved or not. Thisinformation together with the inventors knowledge of the tertiarystructure of subtilisin BPN′ lead the inventors to select a number ofpositions susceptible to mutation with an expectation of obtainingmutants with altered properties. The positions so identified are:¹²⁴Met, ²²²Met, ¹⁰⁴Tyr, ¹⁵²Ala, ¹⁵⁶Glu, ¹⁶⁶Gly, ¹⁶⁹Gly, ¹⁸⁹Phe, ²¹⁷Tyr.Also ¹⁵⁵Asn, ²¹Tyr, ²²Thr, ²⁴Ser, ³²Asp, ³³Ser, ³⁶Asp, ⁴⁶Gly, ⁴⁸Ala,⁴⁹Ser, ⁵⁰Met, ⁷⁷Asn, ⁸⁷Ser, ⁹⁴Lys, ⁹⁵Val, ⁹⁶Leu, ¹⁰⁷Ile, ¹¹⁰Gly, ¹⁷⁰Lys,¹⁷¹Tyr, ¹⁷²Pro, ¹⁹⁷Asp, ¹⁹⁹Met, ²⁰⁴Ser, ²¹³Lys, and ²²¹Ser, whichpositions are identified as being expected to influence variousproperties of the enzyme. Also, a number of mutations are exemplified tosupport these suggestions. In addition to single mutations in thesepositions the inventors also performed a number of multiple mutations.Further the inventors identify ²¹⁵Gly, ⁶⁷His, ¹²⁶Leu, ¹³⁵Leu, and aminoacid residues within the segments 97-103, 126-129, 213-215, and 152-172as having interest, but mutations in any of these positions are notexemplified.

Especially of interest for the purpose of the present invention theinventors of EP 251 446 suggest to substitute ¹⁷⁰Lys (in subtilisinBPN′, type I-S1), specifically they suggest to introduce Glu or Arg forthe original Lys. It appears that the Glu variant was produced and itwas found that it was highly susceptible to autolytic degradation (cf.pages 48, 121, 123 (Table XXI includes an obvious error, but indicates areduction in autolysis half-time from 86 to 13 minutes) and FIG. 32).

EP 260 105 (Genencor) describes modification of certain properties inenzymes containing a catalytic triad by selecting an amino acid residuewithin about 15 Angstroms from the catalytic triad and replace theselected amino acid residue with another residue. Enzymes of thesubtilase type described in the present specification are specificallymentioned as belonging to the class of enzymes containing a catalytictriad. In subtilisins positions 222 and 217 are indicated as preferredpositions for replacement.

Also, Thomas, Russell, and Fersht (1985) Nature 318 375-376 shows thatexchange of ⁹⁹Asp into ⁹⁹Ser in subtilisin BPN′ changes the pHdependency of the enzyme.

In a subsequent article (1987) J. Mol. Biol. 193 803-813, the sameauthors also discuss the substitution of ¹⁵⁶Ser in place of ¹⁵⁶Glu.

Both these mutations are within a distance of about 15 Angstroms fromthe active ⁶⁴His.

In Nature 328 496-500 (1987) Russel and Fersht discuss the results oftheir experiments and present rules for changing pH-activity profiles bymutating an enzyme to obtain changes in surface charge.

WO 88/08028 (Genex) and WO 88/08033 (Amgen) relate to modifications ofamino acid residues in the calcium binding sites of subtilisin BPN′. Theenzyme is said to be stabilized by substituting more negatively chargedresidues for the original ones.

In WO 89/06279 (Novo Nordisk A/S) position 170 is indicated asinteresting and it is suggested to replace the existing residue withTyr. However, no data are given in respect of such a variant. In WO91/00345 (Novo Nordisk A/S) the same suggestion is made, and it is shownthat the Tyr variant of position 170 in subtilisin 309 (type I-S2)exhibits an improved wash performance in detergents at a pH of about 8(variant S003 in Tables III, IV, V, VI, VII, X). The same substitutionin combination with other substitutions in other positions alsoindicates an improved wash performance (S004, S011-S014, S022-S024,S019, S020, S203, S225, S227 in the same Table and Table VII) all inaccordance with the generic concept of said application.

In EP 525 610 (Solvay) it is suggested to improve the stability of theenzyme (a type 1-S2 subtilase closely related to subtilisin PB92)towards ionic tensides by decreasing the hydrophobicity in certainsurface regions thereof. It is consequently suggested to substitute Glnfor the Arg in position 164 (170 if using BPN′ numbering). No variantscomprising this substitution are disclosed in the application.

In WO 94/02618 (Gist-Brocades N.V.) a number of position 164 (170 ifusing BPN′ numbering) variants of the I-S2 type subtilisin PB92 aredescribed. Examples are provided showing substitution of Met, Val, Tyr,Ile, for the original Arg. Wash performance testing in powder detergentsof the variants indicates a slight improvement. Especially for the Ilevariant wash performance tests on cacao an improvement of about 20-30%is indicated. No stability data are provided.

WO 95/30011, WO 95/30010, and WO 95/29979 (Procter & Gamble Company)describe 6 regions, especially position 199-220 (BPN′ numbering), insubtilisin BPN′ and subtilisin 309, which are designed to change (i.e.decrease) the adsorption of the enzyme to surface-bound soils. It issuggested that decreased adsorption by an enzyme to a substrate resultsin better detergent cleaning performance. No specific detergent washperformance data are provided for the suggested variants.

WO 95/27049 (Solvay S.A.) describes a subtilisin 309 type protease withfollowing mutations: N43R+N116R+N117R (BPN′ numbering). Data indicatethe corresponding variant is having improved stability, compared towild-type.

Industrial Applications of Subtilases

Proteases such as subtilisins have found much utility in industry,particularly in detergent formulations, as they are useful for removingproteinaceous stains.

At present at least the following proteases are known to be commerciallyavailable and many of them are marketed in large quantities in manycountries of the world.

Subtilisin BPN′ or Novo, available from e.g. Sigma, St. Louis, U.S.A.

Subtilisin Carlsberg, marketed by Novo Nordisk A/S (Denmark) asALCALASE® and by Gist-Brocades N.V. (Holland) as MAXATASE®.

Both of these belong to subtilase subgroup I-S1

Among the subtilase sub-group l-S2 the following are known to bemarketed.

A Bacillus lentus subtilisin, subtilisin 309, marketed by Novo NordiskA/S (Denmark) as SAVINASE®. A protein engineered variant of this enzymeis marketed as DURAZYM®.

Enzymes closely resembling SAVINASE®, such as subtilisin PB92, MAXACAL®marketed by Gist-Brocades N.V. (a protein engineered variant of thisenzyme is marketed as MAXAPEM®), OPTICLEAN® marketed by Solvay et Cie.and PURAFECT® marketed by Genencor International.

A Bacillus lentus subtilisin, subtilisin 147, marketed by Novo NordiskA/S (Denmark) as ESPERASE®;

To be effective, however, such enzymes must not only exhibit activityunder washing conditions, but must also be compatible with otherdetergent components during detergent production and storage.

For example, subtilisins may be used in combination with other enzymesactive against other substrates, and the selected subtilisin shouldpossess stability towards such enzymes, and also the selected subtilisinpreferably should not catalyse degradation of the other enzymes. Also,the chosen subtilisin should be resistant to the action from othercomponents in the detergent formulation, such as bleaching agents,oxidizing agents, etc., in particular an enzyme to be used in adetergent formulation should be stable with respect to the oxidizingpower, calcium binding properties, and pH conditions rendered by thenon-enzymatic components in the detergent during storage and in the washliquor during wash.

The ability of an enzyme to catalyze the degradation of variousnaturally occurring substrates present on the objects to be cleanedduring e.g. wash is often referred to as its washing ability,washability, detergency, or wash performance. Throughout thisapplication the term wash performance will be used to encompass thisproperty.

The ability of an enzyme to remain active in the presence of othercomponents of a detergent composition prior to being put to use(normally by adding water in the washing process) is usually referred toas storage stability or shelf life. It is often measured as half-life,t_(1/2). We will use the expression storage stability for this propertythroughout this application to encompass this property.

Naturally occurring subtilisins have been found to possess propertieswhich are highly variable in relation to their washing power or abilityunder variations in parameters such as pH. Several of the above marketeddetergent proteases, indeed, have a better performance than thosemarketed about 20 years ago, but for optimal performance each enzyme hasits own specific conditions regarding formulation and wash conditions,e.g. pH, temperature, ionic strength (═I), active system (tensides,surfactants, bleaching agent, etc.), builders, etc.

As a consequence it is found that an enzyme possessing desirableproperties at low pH and low I may be less attractive at more alkalineconditions and high 1, or an enzyme exhibiting fine properties at highpH and high I may be less attractive at low pH, low I conditions.

Also, it has been found that the storage stability differs between theenzymes, but it has further been found that a specific enzyme exhibitslarge variations in storage stability in respect of different detergentformulations, dependent upon a number of parameters, such as pH, pI,bleach system, tensides, etc., and upon the physical state of thedetergent compositions, which may be in powder, dust, or liquid form.Furthermore it may be concentrated or dilute.

The advent and development of recombinant DNA techniques has had aprofound influence in the field of protein chemistry.

Through the application of this technology it is possible now toconstruct enzymes having desired amino acid sequences, and as indicatedabove a fair amount of research has been devoted to designingsubtilisins with altered properties.

Among the proposals the technique of producing and screening a largenumber of mutated enzymes as described in EP 130 756 (Genentech) (USReissue Pat. No. 34,606 (Genencor)) and International patent publ. no.WO 87/05050 (Genex) correspond to a large extend to the classical methodof isolating native enzymes, submit them to classical mutagenesisprograms (using radiation or chemical mutagens) and screen them fortheir properties. The difference Iles in that these methods are moreefficient through the knowledge of the presence of a large number ofvariant enzymes substituted in a specific position.

A subtilisin enzyme typically comprises about 275 amino acid residues.Each residue is capable of being 1 out of 20 possible naturallyoccurring amino acids.

Therefore one very serious draw-back in that procedure is the very largenumber of mutations generated that have to be submitted to a number ofpreliminary screenings to determine their properties.

A procedure as outlined in these patent applications will consequentlyonly be slightly better than the traditional random mutation procedureswhich have been known for years.

The other known techniques relate to changing specific properties, suchas oxidation stability, thermal stability, Ca-stability,transesterification and hydrolysis rate (EP 260 105 (Genencor)),pH-activity profile (Thomas, Russell, and Fersht, supra), and substratespecificity (International patent publ. no. WO 88/07578 (Genentech)).None of these publications relates to changing either the washperformance of enzymes or their storage stability.

International Patent Application no. PCT/DK88/00002 (Novo Nordisk A/S)proposes to use the concept of homology comparison to determine whichamino acid positions should be selected for mutation and which aminoacids should be substituted in these positions in order to obtain adesired change in wash performance.

By using such a procedure the task of screening is reduced drastically,since the number of mutants generated is much smaller, but with thatprocedure it is only foreseen that enzymes exhibiting the combineduseful properties of the parent enzyme and the enzyme used in thecomparison may be obtained.

Thus, as indicated above no relationship has yet been identified betweenwell defined properties of an enzyme such as those mentioned above andthe wash performance and storage stability of an enzyme in variousdetergent compositions.

The problem seems to be that although much research has been directed atrevealing the mechanism of enzyme activity, still only little is knownabout the factors in structure and amino acid residue combination thatdetermine the properties, such as storage stability in detergents, ofenzymes in relation to most of their characteristics, especially whenthe enzymes are present in complex mixtures.

Consequently there still exists a need for further improvement andtailoring of enzymes to detergent systems, as well as a betterunderstanding of the mechanism of protease action and degradation in thepractical use of cleaning or detergent compositions. Such anunderstanding could result in rules which may be applied for selectingmutations that with a reasonable degree of certainty will result in anenzyme exhibiting improved storage stability under specified conditionsin a detergent composition.

SUMMARY OF THE INVENTION

It has now surprisingly been found that a subtilase variant havingimproved storage stability and/or improved performance in detergents,can be obtained by substituting one or more amino acid residues situatedin, or in the vicinity of a hydrophobic domain of the parent subtilasefor an amino acid residue more hydrophobic than the original residue,said hydrophobic domain comprising the residues corresponding toresidues P129, P131, I165, Y167, Y171 of BLS309 (in BASBPN numbering),and said residues in the vicinity thereof comprises residuescorresponding to the residues E136, G159, S164, R170, A194, and G195 ofBLS309 (in BASBPN numbering), with the exception of the R170M, R170I andR170V variants of BABP92.

The present invention relates consequently in its first aspect to enzymevariants exhibiting improved stability and/or improved wash performancein detergent.

In its second aspect the invention relates to DNA constructs capable ofexpressing the enzymes of the first aspect, when inserted in a suitablemanner into a host cell that subsequently is brought to express thesubtilisin enzyme(s) of the first aspect.

In a third aspect the invention relates to the production of thesubtilisin enzymes of the invention by inserting a DNA constructaccording to the second aspect into a suitable host, cultivating thehost to express the desired subtilase enzyme, and recovering the enzymeproduct.

The invention relates, in part, but is not limited to, mutants of thegenes expressing the subtilase sub-group I-S2 enzymes and the ensuingenzyme variants, as indicated above.

Other subtilase gene variants encompassed by the invention are such asthose of the subtilase subgroup I-S1, e.g. subtilisin BPN′, andsubtilisin Carisberg genes and ensuing variant subtilisin BPN′,Proteinase K, and subtilisin Carlsberg enzymes, which exhibit improvedstability in concentrated liquid detergents.

Still further subtilase gene variants encompassed by the invention aresuch as Proteinase K and other genes and ensuing variant Proteinase K,and other subtilase enzymes, which exhibit improved stability inconcentrated liquid detergents.

Other examples of parent subtilase enzymes that can be modified inaccordance with the invention are listed in Table 1.

Further the invention relates to the use of the mutant enzymes incleaning compositions and cleaning compositions comprising the mutantenzymes, especially detergent compositions comprising the mutantsubtilisin enzymes. Specifically the invention relates to concentratedliquid detergent compositions comprising such enzyme variants.

Abbreviations Amino Acids A=Ala=Alanine V=Val=Valine L=Leu=LeucineI=Ile=Isoleucine P=Pro=Proline F=Phe=Phenylalanine W=Trp=TryptophanM=Met=Methionine G=Gly=Glycine S=Ser=Serine T=Thr=ThreonineC=Cys=Cysteine Y=Tyr=Tyrosine N=Asn=Asparagine Q=Gln=GlutamineD=Asp=Aspartic Acid E=Glu=Glutamic Acid K=Lys=Lysine R=Arg=ArginineH=His=Histidine

x=Xaa=Any amino acid

Nucleic Acid Bases A=Adenine G=Guanine C=Cytosine

T=Thymine (only in DNA)U=Uracil (only in RNA)

Variants

In describing the various enzyme variants produced or contemplatedaccording to the invention, the following nomenclatures have beenadapted for ease of reference:

-   -   Original amino acid(s) position(s) substituted amino acid(s)

According to this the substitution of Glutamic acid for glycine inposition 195 is designated as:

-   -   Gly 195 Glu or G195E        a deletion of glycine in the same position is:    -   Gly 195* or G195*        and insertion of an additional amino acid residue such as lysine        is:    -   Gly 195 GlyLys or G195GK

Where a deletion in comparison with the sequence used for the numberingis indicated, an insertion in such a position is indicated as:

-   -   *36 Asp or *36D        for insertion of an aspartic acid in position 36

Multiple mutations are separated by pluses, i.e.:

-   -   Arg 170 Tyr+Gly 195 Glu or R170Y+G195E        representing mutations in positions 170 and 195 substituting        tyrosine and glutamic acid for arginine and glycine,        respectively.

Positions

In describing the variants in this application and in the appendedclaims use is made of the alignment of various subtilases in Siezen etal., supra. In other publications relating to subtilases otheralignments or the numbering of specific enzymes have been used. It is aroutine matter for the skilled person to establish the position of aspecific residue in the numbering used here. Reference is also made toFIG. 1 showing an alignment of residues relevant for the presentinvention from a large number of subtilases. Reference is also made toTable I of WO 91/00345 showing an alignment of residues relevant for thepresent invention from a number of subtilases.

TABLE I Presently established Subtilases (from Siezen et al., supra)cDNA, Organism gene enzyme acronym PROKARYOTES Bacteria: Gram-positiveBacillus subtilis 168 apr A subtilisin I168, apr ABSS168 Bacillusamyloliquefaciens apr subtilisin BASBPN BPN'(NOVO) Bacillus subtilis DY− subtilisin DY BSSDY Bacillus licheniformis + subtilisin CarlsbergBLSCAR Bacillus lentus + subtilisin 147 BLS147 Bacillus alcalophilusPB92 + subtilisin PB92 BAPB92 Bacillus sp. DSM 4828 − alkaline proteaseBDSM48 Bacillus YaB ale alkaline elastase BYSYAB YaB Bacillus subtilis168 epr min. extracell. prot. BSEPR Bacillus subtilis bpfbacillopeptidase F BSBPF Bacillus subtilis IFO3013 ispl intracell.ser.prot.1 BSISP1 Bacillus subtilis A50 − intracell.ser. prot. BSIA50Bacillus thuringiensis − extracell. ser. prot. BTFINI Bacillus cereus −extracell. ser. prot. BCESPR Nocardiopsis dassonvillei − alkaline ser.prot. NDAPII Thermoactinomyces vulgaris − thermitase TVTHER Enterococcusfaecalis cylA cytolysin EFCYLA component A Staphylococcus epidermidisepiP epidermin lead. SEEPIP prot. Streptococcus pyrogenes scpA C5apeptidase SPSCPA Lactococcus lactis SK11 prtP SK11 cell wall prot.LLSK11 Bacteria: Gram-negative Dichelobacter nodosus + basic proteaseDNEBPR Xanthomonas campestris + extracellular prot. XCEXPR Serratiamarcescens + extracell. ser. prot. SMEXSP Thermus aquaticus YT-1 pstlaqualysin I TAAQUA Thermus rT41A + T41A protease TRT41A Vibrioalginolyticus proA protease A VAPROA Streptomyces rutgersensis −proteinase D SRESPD Archaea − halophil extra. prot. ARB172 halophilicstrain 172P1 Cyanobacteria prcA Ca-dependent AVPRCA Anabaena variabilisprotease LOWER EUKARYOTES Fungi Tritirachium album Limber + proteinase KTAPROK Tritirachium album + proteinase R TAPROR Tritirachium album proTproteinase T TAPROT Aspergillus oryzae + alkaline protease AOALPRMalbranchea pulchella − thermomycolin MPTHMY Acremonium chrysogenum alpalkaline protease ACALPR Yeasts Kluyveromyces-lactis kex1 Kex1 ser.proteinase KLKEX1 Saccharomyces cerevisiae kex2 Kex2 ser. proteinaseSCKEX2 Saccharomyces cerevisiae prb1 protease B SCPRB1 Yarrowialipolytica xpr2 alk. extracell. prot. YLXPR2 HIGHER EUKARYOTES WormsCaenorhabditis elegans bli4 cuticle protease CEBLI4 Insects Drosophila(fruit fly) fur1 furin 1 DMFUR1 Drosophila (fruit fly) fur2 furin 2DMFUR2 Plants Cucumis melo (melon) − cucumisin CMCUCU Mammals Human(also rat, mouse) fur furin HSFURI Human (also mouse) + insulinoma PC2HSIPC2 prot. Mouse + pituitary PC3 prot. MMPPC3 Human + tripeptidylpeptid.II HSTPP

REFERENCES USED FOR TABLE I

References to amino acid sequences (GenBank®/EMBL Data Bank accessionnumbers are shown in brackets):

-   ARB172 Kamekura and Seno, (1990) Biochem. Cell Biol. 68, 352-359    (amino acid sequencing of mature protease residues 1-35; residue 14    not determined).-   BSS168 Stahl and Ferrari, (1984) J. Bacteriol. 158, 411-418    (K01988). Yoshimoto, Oyama et al. (1488) J. Biochem. 103, 1060-1065    (the mature subtilisin from B. subtilis var. amylosacchariticus    differs in having T130S and T162S). Svendsen, et al. (1986) FEBS    Lett. 196, 228-232 (PIR A23624; amino acid sequencing; the mature    alkaline mesentericopeptidase From B. mesentericus differs in having    S85A, A88S, S89A, S183A and N259S).-   BASBPN Wells, et al. (1983) Nucl. Acids Res. 11, 7911-7925 (X00165).    Vasantha et al., (1984) J. Bacterol. 159, 811-814 (K02496).-   BSSDY Nedkov et al. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364,    1537-1540 (PIR A00969; amino acid sequencing).-   BLSCAR Jacobs et al. (1985) Nucleic Acids Res. 13, 8913-8926    (X03341). Smith et al., (1968) J. Biol. Chem. 243, 21842191 (PIR    A00968; amino acid sequencing; mature protease sequence differs in    having T103S, P129A, S158N, N161S and S212N).-   BLS147 Hastrup et al. (1989) PCT Patent Appl. WO 89/06279. Pub. Jul.    13, 1989. (Esperase® from B. lentus). Takami et al. (1990) Appl.    Microbiol. Biotechnol., 33, 519-523 (amino acid sequencing of mature    alkaline protease residues 1-20 from Bacillus sp. no. AH-101; this    sequence differs from BLS147 in having N11S).-   BABP92 van der Laan et al. (1991) Appl. Environ. Microbiol. 57    901-909. (Maxacal®). Hastrup et al. (1989) PCT Patent Appl. WO    89/06279. Pub. 13 Jul. 1989. (subtilisin 309). Savinase®, from B.    lentus differs only in having N87S). Godette et al. (1991) Abstracts    5th Protein Society Symposium, June 6, Baltimore: abstract M8 (a    high-alkaline protease from B. lentus differs in having N87S, S99D,    S101R, S103A, V104I and G159S).-   BDSM48 Rettenmaier et al. (1990) PCT Patent Appl. WO 90/04022. Publ.    Apr. 19, 1990.-   BYSYAB Kaneko et al. (1989) J. Bacteriol. 171, 5232-5236 (M28537).-   BSEPR Sloma et al., (1988) J. Bacteriol. 170, 5557-5563 (M22407).    Bruckner (1990) Mol. Gen. Genet. 221, 486-490 (X53307).-   BSBPF Sloma et al., (1990) J. Bacteriol. 172, 1470-1477 (M29035;    corrected). Wu et al. (1990) J. Biol. Chem. 265, 6845-6850 (J05400;    this sequence differs in having A169V and 586 less C-terminal    residues due to a frameshift).-   BSISP1 Koide et al. (1986) J. Bacteriol. 167, 110-116 (M13760).-   BSIA50 Strongin et al. (1978) J. Bacteriol. 133, 1401-1411 (amino    acid sequencing of mature protease residues 1-54; residues 3, 39,    40, 45, 46, 49 and 50 not determined).-   BTFINI Chestukhina et al. (1985) Biokhimiya 50, 1724-1730 (amino    acid sequencing of mature protease residues 1-14 from B.    thuringiensis variety israeliensis, and residues I-16 and 223-243    from variety finitimus). Kunitate et al. (1989) Agric. Biol. Chem.    53, 3251-3256 (amino acid sequencing of mature protease residues    6-20 from variety kurstaki. BTKURS).-   BCESPR Chestukhina et al., (1985) Biokhimiya 50, 1724-1730 (amino    acid sequencing of mature residues I-16 and 223-243).-   NDAPII Tsujibo et al., (1990) Agric. Biol. Chem. 54, 2177-2179    (amino acid sequencing of mature residues 1-26).-   TVTHER Meloun et al. (1985) FEBS Lett. 183, 195-200 (PIR A00973;    amino acid sequencing of mature protease residues 1-274).-   EFCYLA Segarra et al. (1991) Infect. Immun. 59, 1239-1246.-   SEEPIP Schnell et al. (1991) personal communication (Siezen et al.    (supra)).-   SPSCPA Chen et al. (1990) J. Biol. Chem. 265, 3161-3167 (J05224).-   DNEBPR Kortt et al. (1991) Abstracts 5th Protein Society Symposium,    June 22-26, Baltimore, abstract S76.-   LLSK11 Vos et al., (1989) J. Biol. Chem. 264, 13579-13585 (J04962).    Kok et al., (1988) Appl. Environ. Microbiol. 54, 231-238 (M24767;    the sequence from strain Wg2 differs in 44 positions, including 18    differences in the protease domain, and a deletion of residues    1617-1676). Kiwaki et al. (1989) Mol. Microbiol. 3, 359-369 (X14130;    the sequence from strain NCD0763 differs in 46 positions, including    22 in the protease domain, and a deletion of residues 1617-1676).-   XCEXPR Liu et al. (1990) Mol. Gen. Genet. 220, 433-440.-   SMEXSP Yanagida et al. (1986) J. Bacteriol. 166, 937-994 (M13469).-   TAAQUA Terada et al. (1990) J. Biol. Chem. 265, 6576-6581 (J05414).-   TRT41A McHale et al. (1990) Abstracts 5th Eur. Congr. Biotechn.    Christiansen, Munck and Villadsen (eds), Munksgaard Int. Publishers,    Copenhagen.-   VAPROA Deane et al. (1989) Gene 76 281-288 (M25499).-   SRESPD Lavrenova et al. (1984) Biochemistry USSR. 49, 447-454 (amino    acid sequencing of residues 1-23; residues 13, 18 and 19 not    determined).-   AVPRCA Maldener et al. (1991) Mol. Gen. Genet. 225, 113-120 (the    published sequence has 28 uncertain residues near position 200-210    due to a frameshift reading error).-   TAPROK Gunkel and Gassen (1989) Eur. J. Biochem. 179, 185-194    (X14688/XI4689). Jany et al. (1986) J. Biol. Chem. Hoppe-Seyler 367    87(PIR A24541; amino acid sequencing; mature protease differs in    having S745G, SILST204-208DSL and VNLL264-267FNL).-   TAPROR Samal et al. (1990) Mol. Microbiol. 4, 1789-1792 (X56116).-   TAPROT Samal et al., (1989) Gene 85, 329-333.-   AOALPR Tatsumi et al. (1989) Mol. Gen. Genet. 219, 33-38.    Cheevadhanarah et al. (1991) EMBL Data Library (X54726).-   MPTHMY Gaucher and Stevenson (1976) Methods Enzymol. 45, 415-433    (amino acid sequencing of residues 1-28, and hexapeptide LSGTSM with    active site serine).-   ACALPR Isogai et al. (1991) Agric. Biol. Chem. 55, 471-477. Stepanov    et al. (1986) Int. J. Biochem. 18, 369375 (amino acid sequencing of    residues 1-27: the mature protease differs in having H13[1]Q,    R13[2]N and S13[6]A).-   KLKEX1 Tanguy-Rougeau, Wesolowski-Louvel and Fukuhara (1988) FEBS    Lett. 234, 464-470 (X07038).-   SCKEX2 Mizuno et al. (1988) Biochem. Biophys. Res. Commun. 156,    246-254(M24201).-   SCPRB1 Moehle et al. (1987) Mol. Cell. Biol. 7, 43904399 (M18097).-   YLXYPR2Davidow et al., (1987) J. Bacteriol. 169, 4621-4629 (M17741).    Matoba et al., (1988) Mol. Cell. Biol. 8, 4904-4916 (M23353).-   CEBL14 Peters and Rose (1991) The Worm Breeder's Gazette 11, 28.-   DMFUR1Roebroek et al. (1991) FEBS Lett. 289, 133-137 (X59384).-   DMFUR2Roebroek et al. (1992) 267, 17208-17215.-   CMCUCU Kaneda et al. (1984) J. Biochem. 95, 825-829 (amino acid    sequencing of octapeptide NIISGTSM with active site serine).-   HSFURI van den Ouweland et al. (1990) Nucl. Acids Res. 18,664    (X04329) (the sequence of mouse furin differs in 51 positions,    including five in the catalytic domain: A15E, Y21F, S223F, A232V and    N258[2]D). Misumi et al.(1990) Nucl. Acids Res. 18,6719(X55660: the    sequence of rat furin differs in 49 positions, including three in    the catalytic domain: A15E, Y21F, H24R).-   HSIPC2 Smeekens and Steiner (1990) J. Biol. Chem. 265, 2997-3000    (J05252). Seidah et al. (1990) DNA Cell Biol. 9, 415-424 (the    sequence of mouse pituitary PC2 protease differs in 23 positions,    including seven in the protease domain: 14F, S42[2]Y, E45D, N76S,    D133E, V134L and G239[1]D).-   MMPPC3 Smeekens et al., (1991) Proc. Natl. Acad. Sci. USA 88,    340-344 (M58507). Seidah et al., (1990) DNA Cell Biol. 9, 415424    (M55668/M55669; partial sequence).-   HSTPP Tomkinson and Jonsson (1991) Biochemistry 30, 168-174    (J05299).

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show an alignment of a number of the subtilases (SEQ IDNOs: 7-41). For optimal alignments, dashes are shown within thesequences. Further, the numbers on the extreme left designate therespective amino acid sequences as follows:

 1: (BASBPN) SEQ ID NO: 7  2: (ABASS168) SEQ ID NO: 8  3: (BSSDY) SEQ IDNO: 9  4: (BLSCAR) SEQ ID NO: 10  5. (BAPB92) SEQ ID NO: 11  6. (BYSYAB)SEQ ID NO: 12  7. (BLS147) SEQ ID NO: 13  8. (BSEPR) SEQ ID NO: 14  9.(BSISP1) SEQ ID NO: 15 10. (TVTHER) SEQ ID NO: 16 11. (DNEBPR) SEQ IDNO: 17 12. (XCEXPR) SEQ ID NO: 18 13. (BSBPF) SEQ ID NO: 19 14. (EFCYLA)SEQ ID NO: 20 15. (SEEPIP) SEQ ID NO: 21 16. (SPSCPA) SEQ ID NO: 22 17.(LLSK11) SEQ ID NO: 23 18. (SMEXSP) SEQ ID NO: 24 19. (AVPRCA) SEQ IDNO: 25 20. (MMPPC3) SEQ ID NO: 26 21. (HSIPC2) SEQ ID NO: 27 22.(HSFURI) SEQ ID NO: 28 23. (DMFUR1) SEQ ID NO: 29 24. (KLKEX1) SEQ IDNO: 30 25. (SCKEX2) SEQ ID NO: 31 26. (VAPROA). SEQ ID NO: 32 27.(TRT41A) SEQ ID NO: 33 28. (TAAQUA) SEQ ID NO: 34 29. (TAPROK) SEQ IDNO: 35 30. (TAPROR) SEQ ID NO: 36 31. (TAPROT) SEQ ID NO: 37 32.(ACALPR) SEQ ID NO: 38 33. (AOALPR) SEQ ID NO: 39 34. (SCPRB1) SEQ IDNO: 40 35. (YLXPR2). SEQ ID NO: 41

FIG. 2 is a 3 dimensional representation of subtilisin 309 showing thelocation of the hydrophobic domain and some of the amino acid residuesin the vicinity thereof to be substituted according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

It has surprisingly been found that the storage stability and/orimproved performance in detergents of subtilases generally is improvedwhen amino acid residues situated in, or in the vicinity of ahydrophobic domain comprising the residues P129, P131, I165, Y167, Y171of subtilisin 309 are substituted for a more hydrophobic residue. Theresidues in question are especially E136, G159, S164, R170, A194, andG195.

Further, said variant exhibits a particularly high improved stability inliquid detergents and in detergents in a shaped solid form.

FIG. 2 shows the hydrophobic domain in subtilisin 309 and residues inthe vicinity thereof a number of which are to be substituted in order toincrease the hydrophobicity of the domain. This may be achieved bysubstituting hydrophobic residues for non-hydrophobic residues and/or bysubstituting residues to become even more hydrophobic than in the parentenzyme.

The same principle applies to the corresponding domain in othersubtilases, the identification of which is within the skills of theaverage person working in this technical field. Graphic representationslike the one in FIG. 2 can be produced for other subtilases to determinethe target residues to be substituted according to the invention.

A number hereof is indicated in Table II below:

TABLE II residues in hydrophobic domain and the vicinity thereof Enz.Pos BASBPN BLSCAR BLS309 BLS147 TVTHER domain 129 P A P T T 131 G G P GG 165 V I I V P 167 Y Y Y Y Y 171 Y Y Y Y Y Vicinity 136 K K E E Q 159 SS G G T 164 T T S G A 170 K K R R Y 194 P A A P S 195 E E G E V

Table II was constructed using the alignment shown in FIG. 2. It isobvious that similar or larger tables covering other subtilases mayeasily be produced by the skilled person.

Consequently the invention relates to subtilase variants in which theamino acid sequence has been changed through mutating the gene of thesubtilisin enzyme, which it is desired to modify (the parent enzyme orgene), in the codon responsible for the expression of the amino acidresidue in positions 129, 131, 165, 167, 171, 136, 159, 164, 170, 194,and 195, which residues are more hydrophobic than the residue(s) in theparent enzyme, especially such hydrophobic residues that comprise arelatively long hydrophobic side chain, such as Ile, Leu, and Val,whereby, when the mutated gene is expressed, the amino acid residue issubstituted by a more hydrophobic residue, which increases thehydrophobicity of the domain as such.

Hydrophobic amino acid residues are generally the following:

Val (V), Ile (I), Leu (L), Met (M), Phe (F), Pro (P) and Trp (W). Amongthese Val, Ile and Leu are preferred.

By looking at Table II and applying the principle of the invention anumber of candidates for substitution becomes clear.

For both BASBPN and BLSCAR it seems appropriate to make substitutions inpositions 129, 131, 136, 159, 164, 167, 170, 171 and 195. In BLS309positions 136, 164, 167, and 170, 171 would be the first choices, andpositions 159 and 195 also would be a second choice. In BLS147 positions129, 131, 136, 167, 170, 171 and 195 are the first choice, whilepositions 159 and 164 are second. Finally, in TVTHER positions 129, 131,136, 167, 171 and 194 are the first choices, with 164 as a second one.

According to the invention it would entail an advantage to substitutethe Gly residues in the hydrophobic domain to bulkier and morehydrophobic residues.

Such considerations apply for any hydrophilic or hydrophobic residuethat may occupy any of the above mentioned position, meaning that anyincrease in hydrophobicity seems to be advantageous. This means thate.g. a very hydrophilic residue such as the charged residues Arg (R),Asp (D), Glu (E) or Lys (K) may be substituted by any residue that isless hydrophilic. Such less hydrophilic residues comprises the residuesGly (G), Cys (C), Ser (S), Ala (A), Thr (T), Tyr (Y), Gin (O), His (H)or Asn (N). It also means that a Tyr(Y) may be substituted by a morehydrophobic residue such as Phe(F), Leu(L), or Ile(I).

Similar considerations can be applied to other subtilases having ahydrophobic domain in this part of the surface of the enzyme.

In the context of this invention a subtilase is defined in accordancewith Siezen et al., supra. In a more narrow sense, applicable to manyembodiments of the invention, the subtilases of interest are thosebelonging to the subgroups I-S1 and I-S2. In a more specific sense, manyof the embodiments of the invention relate to serine proteases ofgram-positive bacteria which can be brought into substantiallyunambiguous homology in their primary structure, with the subtilaseslisted in Table I above.

The present invention also comprises any one or more substitutions inthe above mentioned positions in combination with any othersubstitution, deletion or addition to the amino acid sequence of theparent enzyme. Especially combinations with other substitutions known toprovide improved properties to the enzyme are envisaged.

Such combinations comprise the positions: 222 (improve oxidationstability), 218 (improves thermal stability), substitutions in theCa-binding sites stabilising the enzyme, e.g. position 76, and manyother apparent from the prior art.

Furthermore combinations with the variants mentioned in EP 405 901 arealso contemplated specifically.

Variants A: Single Variants: Subtilisin BPN′, Subtilisin Carlsberg,Subtilisin 168, and Subtilisin DY Variants:

A129V, A129I, A129L, A129M, A129F,

G131V, G131I, G131L, G131M, G131F,

K136V, K136I, K136L, K136M, K136F,

S159V, S159I, S159L, S159M, S159F,

T164V, T164I, T164L, T164M, T164F,

Y167V, Y167I, Y167L, Y167M, Y167F,

K170V, K170I, K170L, K170M, K170F,

Y171V, Y171I, Y171L, Y171M, Y171F,

A194V, A194I, A194L, A194M, A194F,

E195V, E195I, E195L, E195M, E195F,

Thermitase Variants:

A129V, A129I, A129L, A129M, A129F,

G131V, G131I, G131L, G131M, G131F,

Q136V, Q136I, Q136L, Q136M, Q136F,

T159V, T159I, T159L, T159M, T159F,

A164V, A164I, A164L, A164M, A164F,

Y167V, Y167I, Y167L, Y167M, Y167F,

Y171V, Y171I, Y171L, Y171M, Y171F,

Y170V, Y170I, Y170L, Y170M, Y170F,

S194V, S194I, S194L, S194M, S194F,

Subtilisin 309, Subtilisin 147, and Bacillus PB92 Protease Variants:

T129V, T129I, T129L, T129M, T129F,

G131V, G131I, G131L, G131M, G131F,

E136V, E136I, E136L, E136M, E136F,

G159V, G159I, G159L, G159M, G159F,

G164V, G164I, G164L, G164M, G164F, (BLS147)

S164V, S164I, S164L, S164M, S164F, (BLS309 AND BAPB92)

Y167A, Y167H, Y167N, Y167P, Y167C, Y167W, Y167Q, Y167S, Y167T, Y167G,Y167V, Y167I, Y167L, Y167M, Y167F

R170W, R170A, R170H, R170N, R170P, R170Q, R170S, R170T, R170Y(disclaimed for BLS309), R170V (disclaimed for BAPB92), R170I(disclaimed for BAPB92),

R170L, R170M (disclaimed for BAPB92), R170F, R170G, R170C,

Y171A, Y171H, Y171N, Y171P, Y171C, Y171W, Y171Q, Y171S, Y171T, Y171G,Y171V, Y171I, Y171L, Y171M, Y171F,

A194V, A194I, A194L, A194M, A194F, (BLS309 AND BAPB92)

P194V, P194I, P194L, P194M, P194F, (BLS147)

E195V, E195I, E195L, E195M, E195F, (BLS147)

G195V, G195I, G195L, G195M, G195F, (BLS309 AND BAPB92

B: Combination Variants:

Any of the above variants are contemplated to prove advantageous ifcombined with other variants in any of the positions:

27, 36, 57, 76, 97, 101, 104, 120, 123, 206, 218, 222, 224, 235 and 274.

Specifically the following BLS309 and BAPB92 variants are consideredappropriate for combination: K27R, *36D, S57P, N76D, G97N, S101G, V104A,V104N, V104Y, H120D, N123S, A194P, Q206E, N218S, M222A, M222S, T224S,K235L and T274A.

Also such variants comprising any one or two of the substitutions X167F,X167I, X167L, X167M, X167V, X170F, X170I, X170L, X170M, and/or X170V, incombination with any one or more of the other substitutions, deletionsand/or insertions mentioned above are advantageous.

Furthermore variants comprising any of the variants V104N+S101G,K27R+V104Y+N123S+T274A, or N76D+V104A or other combinations of thesemutations (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, V104A), incombination with any one or more of the substitutions, deletions and/orinsertions mentioned above are deemed to exhibit improved properties.

Specific combinations to be mentioned are:

a) S57P+R170L

a′) S57P+R170I

b) R170L+N218S

b′) R170I+N218S

c) S57P+R170L+N218S

c′) S57P+R170I+N218Sc″) S57P+V104Y+R170L+N218Sc″) S57P+V104Y+R170I+N218S

d) R170L+N218S+M222A

d′) R170I+N218S+M222Sd″) R170L+N218S+M222Ad′″) R170I+N218S+M222S

e) S57P+R170L+S188P+A194P

e′) S57P+R170I+S188P+A194P

f) Y167L+R170L f) Y167L+R170I g) Y167I+R170L

g′) Y167I+R170I

h) N76D+R170L+N218S

h′) N76D+R170I+N218S

i) S57P+N76D+R170L+N218S

i′) S57P+N76D+R170I+N218S

j) N76D+R170L+N218S+M222A

j′) N76D+R170I+N218S+M222Sj″) N76D+R1 70L+N218S+M222Aj′″) N76D+R170L+N218S+M222S

k) S57P+R170I+S188P+A194P+N218S

k′) S57P+R170I+S188P+A194P+N218S

l) *36D+N76D+H120D+R170L+G195E+K235L

l′) *36D+N76D+H120D+R170I+G195E+K235Ll″) *36D+N76D+H120D+Y167I+R170L+G195E+K235Ll′″) *36D+N76D+H120D+Y167I+R170I+G195E+K235L

m) N76D+H120D+R170L+G195E+K235L

m′) N76D+H120D+R170I+G195E+K235Lm″) N76D+H120D+Y167I+R170L+G195E+K235Lm′) N76D+H120D+Y167I+R170I+G195E+K235L

n) *36D+G97N+V104Y+H120D+R170L+A194P+G195E+K235L

n′) *36D+G97N+V104Y+H120D+R170I+A194P+G195E+K235L

o) S57P+R170L+Q206E

o′) S57P+R170I+Q206E

p) R170L+Q206E

p′) R170I+Q206E

q) Y167I+R170L+Q206E

q′) Y167I+R170I+Q206E

r) Y167F+R170L

r′) Y167F+R170I

t) Y167I+R170L+A194P

t′) Y167I+R170I+A194Pt″) Y167L+R170L+A194Pt′″) Y167L+R170I+A194P

u) Y167I+R170L+N218S

u′) Y167I+R170I+N218Su″) Y167L+R170L+N218Su′″) Y167L+R170I+N218S

v) Y167I+R170L+A194P+N218S

v′) Y167I+R170I+A194P+N218Sv″) Y167L+R170L+A194P+N218Sv′″) Y167L+R170I+A194P+N218S

x) R170L+P131V

x′) R170I+P131V

y) *36D+Y167I+R170L

y′) *36D+Y167I+R170I

z) Y167I+Y171I aa) Y167V+R170L

aa′) Y167V+R170I

bb) R170L+Y171I

bb′) R170I+Y171Lbb″) R170L+Y171Lbb′″) R170I+Y171I

cc) Y167I+Y171L+N218S

cc′) Y167I+Y171I+N218S

Detergent Compositions Comprising the Mutant Enzymes

The present invention also comprises the use of the mutant enzymes ofthe invention in cleaning and detergent compositions and suchcompositions comprising the mutant subtilisin enzymes. Such cleaning anddetergent compositions can in principle have any physical form, but thesubtilase variants are preferably incorporated in liquid detergentcompositions or in detergent compositions in the form of bars, tablets,sticks and the like for direct application, wherein they exhibitimproved enzyme stability or performance.

Among the liquid compositions of the present invention are aqueousliquid detergents. having for example a homogeneous physical character,e.g. they can consist of a micellar solution of surfactants in acontinuous aqueous phase, so-called isotropic liquids.

Alternatively, they can have a heterogeneous physical phase and they canbe structured, for example they can consist of a dispersion of lamellardroplets in a continuous aqueous phase, for example comprising adeflocculating polymer having a hydrophilic backbone and at least onehydrophobic side chain, as described in EP-A-346 995 (Unilever)(incorporated herein by reference). These latter liquids areheterogeneous and may contain suspended solid particles such asparticles of builder materials e.g. of the kinds mentioned below.

Concerning powder detergent compositions such compositions comprise inaddition to any one or more of the subtilisin enzyme variants inaccordance to any of the preceding aspects of the invention alone or incombination any of the usual components included in such compositionswhich are well-known to the person skilled in the art.

Such components comprise builders, such as phosphate or zeolitebuilders, surfactants, such as anionic, cationic, non-ionic orzwitterionic type surfactants, polymers, such as acrylic or equivalentpolymers, bleach systems, such as perborate- or amino-containing bleachprecursors or activators, structurants, such as silicate structurants,alkali or acid to adjust pH, humectants, and/or neutral inorganic salts.

Furthermore, a number of other ingredients are normally present in thecompositions of the invention, such as Cosurfactants, Tartrate SuccinateBuilder, Neutralization System, Suds Suppressor, Other Enzymes and OtherOptional Components.

The weight ratio of anionic surfactant to nonionic surfactant ispreferably from 1:1 to 5:1. The compositions have a pH in a 10% byweight solution in water at 20° C. of from 7.0 to 9.0, a CriticalMicelle Concentration of less than or equal to 200 ppm, and an air/waterInterfacial Tension at the Critical Micelle Concentration of less thanor equal to 32 dynes/cm at 35° C. in distilled water. The compositionsare preferably clear, homogeneous and phase stable, and have goodcleaning performance and enzyme stability.

Various Components: 1. Anionic Surfactant

The compositions of the present invention contain from about 10% toabout 50%, preferably from about 15% to about 50%, more preferably fromabout 20% to 40%, and most preferably from 20% to about 30%, by weightof a natural or synthetic anionic surfactant. Suitable natural orsynthetic anionic surfactants are e.g. soaps and such as disclosed inU.S. Pat. Nos. 4,285,841 and 3,929,678.

Useful anionic surfactants include the water-soluble salts, particularlythe alkali metal, ammonium and alkylolammonium (e.g.,monoethanolammonium or triethanolammonium) salts, of organic sulfuricreaction products having in their molecular structure an alkyl groupcontaining from about 10 to about 20 carbon atoms and a sulfonic acid orsulfuric acid ester group. (Included in the term “alkyl” is the alkylportion of aryl groups.) Examples of this group of synthetic surfactantsare the alkyl sulfates, especially those obtained by sulfating thehigher alcohols (C₈-C₁₈ carbon atoms) such as those produced by reducingthe glycerides of tallow or coconut oil; and the alkylbenzene sulfonatesin which the alkyl group contains from about 9 to about 15 carbon atoms,in straight chain or branched chain configuration, e.g., those of thetype described in U.S. Pat. Nos. 2,220,099 and 2,477,383. Especiallyvaluable are linear straight chain alkylbenzene sulfonates in which theaverage number of carbon atoms in the alkyl group is from about 11 to14.

Other anionic surfactants herein are the water-soluble salts of:paraffin sulfonates containing from 8 to about 24 (preferably about 12to 18) carbon atoms; alkyl glyceryl ether sulfonates, especially thoseethers of C₈-C₁₈ alcohols (e.g., those derived from tallow and coconutoil); alkyl phenol ethylene oxide ether sulfates containing from 1 toabout 4 units of ethylene oxide per molecule and from 8 to 12 carbonatoms in the alkyl group; and alkyl ethylene oxide ether sulfatescontaining 1 to 4 units of ethylene oxide per molecule and from 10 to 20carbon atoms in the alkyl group.

Other useful anionic surfactants include the water-soluble salts ofesters of alpha-sulfonated fatty.acids containing from 6 to 20 carbonatoms in the fatty acid group and from 1 to 10 carbon atoms in the estergroup; water-soluble salts of 2-acyloxy-alkane-1-sulfonic acidscontaining from 2 to 9 carbon atoms in the acyl group and from 9 to 23carbon atoms in the alkane moiety; water-soluble salts of olefinsulfonates containing from 12 to 24 carbon atoms; and beta-alkyloxyalkane sulfonates containing from 1 to 3 carbon atoms in the alkyl groupand from 8 to 20 carbon atoms in the alkane moiety.

Preferred anionic surfactants are soaps, the C₁₀-C₁₈ alkyl sulfates andalkyl ethoxy sulfates containing an average of up to 4 ethylene oxideunits per mole of alkyl sulfate, C₁₁-C₁₃ linear alkyl benzenesulfonates, and mixtures thereof.

2. Nonionic Surfactant

Another optional ingredient is from 2% to 14% preferably from 2% to 8%,most preferably from 3% to 5% by weight, of an optionally ethoxylatednonionic surfactant. The weight ratio of natural or synthetic anionicsurfactant (on an acid basis) to nonionic surfactant is from 1:1 to 5:1preferably from 2:1 to 5:1, most preferably from 3:1 to 4:1. This is toensure the formation and adsorption of sufficient hardness surfactantsat the air/water interface to provide good greasy/oily soil removal.

The optionally ethoxylated nonionic surfactant is of the formulaR¹(OC₂H₄)_(n)OH, wherein R¹ is a C₁₀-C₁₆ alkyl group or a C₈-C₁₂ alkylphenyl group, n is from 3 to 9, and said nonionic surfactant has an HLB(Hydrophilic-Lipophilic Balance) of from 6 to 14, preferably from 10 to13. These surfactants are more fully described in U.S. Pat. Nos.4,285,841 and 4,284,532. Particularly preferred are condensationproducts of C₁₂-C₁₅ alcohols with from 3 to 8 moles of ethylene oxideper mole of alcohol, e.g., C₁₂-C₁₃ alcohol condensed with about 6.5moles of ethylene oxide per mole of alcohol. Other nonionic surfactantsto be mentioned are APG, EGE, and glucamide surfactants.

3. Detergency Builder

Among the usual detergent ingredients which may be present in usualamounts in the detergent compositions of this invention are thefollowing: The compositions may be built or unbuilt, and may be of thezero-P type (i.e. not containing any phosphorus containing builders).Thus, the composition may contain in the aggregate for example from1-50%, e.g. at least about 5% and often up to about 35-40% by weight, ofone or more organic and/or inorganic builders. Typical examples ofbuilders include those already mentioned above, and more broadly includealkali metal ortho, pyro, and tripolyphosphates, alkali metalcarbonates, either alone or in admixture with calcite, alkali metalcitrates, alkali metal nitrilotriacetates, carboxymethyloxysuccinates,zeolites, polyacetalcarboxylates, and so on.

More specifically the compositions herein contain from 5% to 20%,preferably from 10% to 15%, by weight of a detergency builder which canbe a fatty acid containing from 10 to 18 carbon atoms and/or apolycarboxylate, zeolite, polyphoshonate and/or polyphosphate a builder.Preferred are from 0 to 10% (more preferably from 3% to 10%) by weightof saturated fatty acids containing from 12 to 14 carbon atoms, alongwith from 0 to 10%, more preferably from 2% to 8%, most preferably from2% to 5%, by weight of a polycarboxylate builder, most preferably citricacid, in a weight ratio of from I:I to 3:1.

Since the proteolytic enzymes herein appear to provide optimum storagestability benefits versus other enzymes when the builder to waterhardness ratio is close to one, the compositions preferably containsufficient builder to sequester from 2 to 10, preferably from 3 to 8,grains per gallon of hardness.

Suitable saturated fatty acids can be obtained from natural sources suchas plant or animal esters (e.g., palm kernel oil, palm oil and coconutoil) or synthetically prepared (e.g., via the oxidation of petroleum orby hydrogenation of carbon monoxide via the Fisher-Tropsch process).Examples of suitable saturated fatty acids for use in the compositionsof this invention include capric, lauric, myristic, coconut and palmkernel fatty acid. Preferred are saturated coconut fatty acids; from 5:1to I:I (preferably about 3:1) weight ratio mixtures of lauric andmyristic acid; mixtures of the above with minor amounts (e.g., 1%-30% oftotal fatty acid) of oleic acid; and palm kernel fatty acid.

The compositions herein preferably also contain the polycarboxylate,polyphosphonate and polyphosphate builders described in U.S. Pat. No.4,284,532, Water-soluble polycarboxylate builders, particularlycitrates, are preferred of this group. Suitable polycarboxylate buildersinclude the various aminopolycarboxylates, cycloalkane polycarboxylates,ether polycarboxylates, alkyl polycarboxylates, epoxy polycarboxylates,tetrahydrofuran polycarboxylates, benzene polycarboxylates, andpolyacetal polycarboxylates.

Examples of such polycarboxylate builders are sodium and potassiumethylenediaminetetraacetate; sodium and potassium nitrilotriacetate; thewater-soluble salts of phyfic acid, e.g., sodium and potassium phytates,disclosed in U.S. Pat. No. 1,739,942, the polycarboxylate materialsdescribed in U.S. Pat. No. 3,364,103; and the water-soluble salts ofpolycarboxylate polymers and copolymers described in U.S. Pat. No.3,308,067.

Other useful detergency builders include the water-soluble salts ofpolymeric aliphatic polycarboxylic acids having the following structuraland physical characteristics: (a) a minimum molecular weight of about350 calculated as to the acid form; (b) an equivalent weight of 50 to 80calculated as to acid form; (3) at least 45 mole percent of themonomeric species having at least two carboxyl radicals separated fromeach other by not more than two carbon atoms: (d) the site of attachmentof the polymer chain of any carboxyl-containing radical being separatedby not more than three carbon atoms along the polymer chain from thesite of attachment of the next carboxyl-containing radical. Specificexamples of such builders are the polymers and copolymers of itaconicacid, aconitic acid, maleic acid, mesaconic acid, fumaric acid,methylene malonic acid, and citraconic acid.

Other suitable polycarboxylate builders include the water-soluble salts,especially the sodium and potassium salts, of mellitic acid, citricacid, pyromellitic acid, benzene pentacarboxylic acid, oxydiacetic acid,carboxymethyloxy-succinic acid, carboxymethyloxymalonic acid,cis-cyclohexanehexacarboxylic acid, cis-cyclopentanetetracarboxylic acidand oxydisuccinic acid.

Other polycarboxylates are the polyacetal carboxylates described in U.S.Pat. Nos. 4,144,226, and 4,146,495.

Other detergency builders include the zeolites, such as thealuminosilicate ion exchange material described in U.S. Pat. No.4,405,483.

Other preferred builders are those of the general formulaR—CH(COOH)CH₂(COOH), i.e. derivatives of succinic acid, wherein R isC₁₀-C₂₀ alkyl or alkenyl, preferably C₁₂-C₁₆, or wherein R may besubstituted with hydroxyl, sulfo, sulfoxy or sulfone substituents. Thesesuccinate builders are preferably used in the form of their watersoluble salts, including the sodium, potassium and alkanolammoniumsalts. Specific examples of succinate builders include: laurylsuccinate, myristyl succinate, palmityl succinate, 2-dodecenylsuccinate, and the like.

4. Proteolytic Enzyme

The enzymes of the invention can be used in well-known standard amountsin detergent compositions. The amounts may range very widely, e.g. about0.0002-0.1, e.g. about 0.005-0.05, Anson units per gram of the detergentcomposition. Expressed in alternative units, the protease can beincluded in the compositions in amounts in the order of from about 0.1to 100 GU/mg (e.g. 1-50, especially 5-20 GU/mg) of the detergentformulation, or any amount in a wide range centering at about 0.01-4,e.g. 0.1-0.4 KNPU per g of detergent formulation.

It may for example be suitable to use the present enzymes at the rate ofabout 0.25 mg of enzyme protein per liter of wash liquor, correspondingto an enzyme activity of the order of 0.08 KNPU per liter. Correspondingdetergent formulations can contain the enzymes in for example an amountof the order of 0.1-0.4 KNPU/g.

Expressed differently the compositions of the present invention containfrom about 0.01% to about 5%, preferably from about 0.1% to about 2%, byweight of the proteolytic enzymes of the invention.

The described proteolytic enzyme is preferably included in an amountsufficient to provide an activity of from 0.05 to about 1.0, morepreferably from about 0.1 to 0.75, most preferably from about 0.125 toabout 0.5 mg of active enzyme per gram of composition.

The enzyme component may be added to the other components in anyconvenient form, such as in the form of a solution, slurry, LDP slurry,or crystals.

5. Enzyme Stabilization System

The liquid detergents according to the present invention may comprise anenzyme stabilization system, comprising calcium ion, boric acid,propylene glycol and/or short chain carboxylic acids. The enzymestabilization system comprises from about 0.5% to about 15% by weight ofthe composition.

The composition preferably contains from about 0.01 to about 50,preferably from about 0.1 to about 30, more preferably from about 1 to20 millimoles of calcium ion per liter. The level of calcium ion shouldbe selected so that there is always some minimum level available for theenzyme, after allowing for complexation with builders etc. in thecomposition. Any water-soluble calcium salt can be used as the source ofcalcium ion, including calcium chloride, calcium formate, and calciumacetate. A small amount of calcium ion, generally from about 0.05 to 0.4millimoles per liter, is often also present in the composition due tocalcium in the enzyme slurry and formula water. From about 0.03% toabout 0.6% of calcium formate is preferred.

A second preferred enzyme stabilizer is polyols containing only carbon,hydrogen and oxygen atoms. They preferably contain from 2 to 6 carbonatoms and from 2 to 6 hydroxy groups. Examples include propylene glycol(especially 1,2-propanediol, which is preferred), ethylene glycol,glycerol, sorbitol, mannitol, and glucose. The polyol generallyrepresents from about 0.5% to 15%, preferably from about 1.5% to about8%, by weight of the composition. Preferably, the weight ratio of polyolto any boric acid added is at least 1, more preferably at least 1.3.

The compositions preferably also contain the water-soluble, short chaincarboxylates described in U.S. Pat. No. 4,318,818. The formates arepreferred and can be used at levels of from about 0.05% to about 5%,preferably from about 0.2% to about 2%, most preferably from 0.4% to1.5%, by weight of the composition. Sodium formate is preferred.

The compositions herein also optionally contain from about 0.25% toabout 5%, most preferably from about 0.5% to about 3%, by weight ofboric acid. The boric acid may be, but is preferably not, formed by acompound capable of forming boric acid in the composition. Boric acid ispreferred, although other compounds such as boric oxide, borax and otheralkali metal borates (e.g., sodium ortho-, meta- and pyroborate, andsodium pentaborate) are suitable. [Substituted boric acids (e.g.,phenylboronic acid, butane boronic acid, and p-bromo phenylboronic acid)can also be used in place of boric acid.

6. Water

The liquid compositions of the present invention may be aqueous liquidsor non-aqueous liquids. When the are aqueous liquids, they contain fromabout 15% to about 60%, preferably from about 25% to about 45%, byweight of water.

Further Optional Components A. Cosurfactants

Optional cosurfactants for use with the above nonionic surfactantsinclude amides of the formula

wherein R¹ is an alkyl, hydroxyalkyl or alkenyl radical containing from8 to 20 carbon atoms, and R² and R³ are selected from the groupconsisting of hydrogen, methyl, ethyl, propyl, isopropyl,2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, and said radicalsadditionally containing up to 5 ethylene oxide units, provided at leastone of R² and R³ contains a hydroxyl group.

Preferred amides are the C₈-C₂₀ fatty acid alkylol amides in which eachalkylol group contains from 1 to 3 carbon atoms, and additionally cancontain up to 2 ethylene oxide units. Particularly preferred are theC₁₂-C₁₆ fatty acid monoethanol and diethanol amides.

If used, amides are preferably present at a level such that the aboveethoxylated nonionic surfactant and amide surfactant is in a weightratio of from 4:1 to 1:4, preferably from 3:1 to 1:3.

Preferred and optional cosurfactants, used at a level of from 0.15% to1%, are the quaternary ammonium, amine and amine oxide surfactantsdescribed in U.S. Pat. No. 4,507,219.

Of the above, the C₁₀-C₁₄ alkyl trimethylammonium salts are preferred,e.g., decyl trimethylammonium methylsulfate, lauryl trimethylammoniumchloride, myristyl trimethylammonium bromide, and coconuttrimethylammonium chloride and methylsulfate. From 0.2% to 0.8% ofmonoalkyl trimethylammonium chloride is preferred.

B. Tartrate Succinate Builder

The compositions herein preferably contain from 0 to about 10%,preferably from 0 to about 6%, by weight on an acid basis, of a tartratesuccinate builder material selected from the group consisting of:

wherein X is a salt-forming cation;

wherein X is a salt-forming cation; andiii) mixtures thereof.

The tartrate succinate compounds used herein are described in U.S. Pat.No. 4,663,071.

C. Neutralization System

The present compositions can also optionally contain from about 0 toabout 0.04 moles, preferably from about 0.01 to 0.035 moles, morepreferably from about 0.015 to about 0.03 moles, per 100 grams ofcomposition of an alkanolamine selected from the group consisting ofmonoethanolamine, diethanolamine, triethanolamine, and mixtures thereof.Low levels of the alkanolamines, particularly monoethanolamine, arepreferred to enhance product stability, detergency performance, andodour. However, the amount of alkanolamine should be minimized for bestchlorine bleach compatibility.

In addition, the compositions contain sodium ions, and preferablypotassium ions, at a level sufficient to neutralize the anionic speciesand provide the desired product pH.

D. Suds Suppressor

Another optional component for use in the liquid detergents herein isfrom 0 to about 1.5%, preferably from about 0.5% to about 1.0%, byweight of silicone based suds suppressor agent.

Silicones are widely known and taught for use as highly effective sudscontrolling agents. For example, U.S. Pat. No. 3,455,839 relates tocompositions and processes for defoaming aqueous solutions byincorporating therein small amounts of polydimethylsiloxane fluids.

Useful suds controlling silicones are mixtures of silicone and silanatedsilica as described, for instance, in German Patent Application DOS2,124,526.

Silicone defoamers and suds controlling agents have been successfullyincorporated into granular detergent compositions by protecting themfrom detergent surfactants as in U.S. Pat. Nos. 3,933,672 and 4,652,392.

A preferred silicone based suds suppressor for use herein is a sudssuppressing amount of a suds controlling agent consisting essentiallyof:

(i) polydimethylsiloxane fluid having a viscosity of from about 20 cs.to about 1500 cs. at 25° C.;

(ii) from about 5 to about 50 parts per 100 parts by weight of (i) ofsiloxane resin composed of (CH₃)₃SiO_(1/2) units and SiO₂ units in aratio of from (CH₃)₃SiO_(1/2) units and to SiO₂ units of from about0.6:1 to about 1.2:1; and

(iii) from about 1 to about 20 parts per 100 parts by weight of (i) of asolid silica gel.

By “suds suppressing amount” is meant that the formulator of thecomposition can select an amount of this suds controlling agent thatwill control the suds to the extent desired. The amount of suds controlwill vary with the detergent surfactant selected. For example, with highsudsing surfactants, relatively more of the suds controlling agent isused to achieve the desired suds control than with low foamingsurfactants.

E. Other Enzymes

The detergent compositions of the invention may also contain furtherenzymes. For example, lipase can usefully be added in the form of asolution or a slurry of lipolytic enzyme with carrier material (e.g. asin EP 258 068 (Novo Nordisk A/S)).

The added amount of lipase can be chosen within wide limits, for example50 to 30,000 LU/g per gram of the surfactant system or of the detergentcomposition, e.g. often at least 100 LU/g, very usefully at least 500LU/g, sometimes preferably above 1000, above 2000 LU/g or above 4000LU/g or more, thus very often within the range of 50-4000 LU/g, andpossibly within the range of 200-1000 LU/g. In this specification,lipase units are defined as they are in EP 258 068.

The lipolytic enzyme can be chosen among a wide range of lipases. Inparticular, the lipases described in for example the following patentspecifications: EP 214 761 (Novo Nordisk A/S), 258 068, and especiallylipases showing immunological cross reactivity with antisera raisedagainst lipase from Thermomyces lanuginosus ATCC 22070, EP 205 208 and206 390, and especially lipases showing immunological cross-reactivitywith antisera raised against lipase from Chromobacter viscosum varlipolyticum NRRL B-3673, or against lipase from Alcaligenes PL-679, ATCC31371 and FERM-P 3783, also the lipases described in WO 87/00859(Gist-Brocades) and EP 204 284 (Sapporo Breweries). Suitable, inparticular, are for example the following commercially available lipasepreparations: Lipolase® Novo Nordisk A/S, Amano lipases CE, P, B, AP,M-AP, AML, and CES, and Meito lipases MY-30, OF, and PL, also Esterase®MM (Novo Nordisk A/S), Lipozym, SP225, SP285, (all Novo Nordisk A/S)Saiken lipase, Enzeco lipase, Toyo Jozo lipase and Diosynth lipase(Trade Marks), Lumafast® (Genencor Inc.), Lipomax® (Gist-Brocades N.V.),and lipases as described in WO 94/03578 (Unilever).

Amylase can for example be used when desired, in an amount in the rangeof about 1 to about 100 MU (maltose units) per gram of detergentcomposition (or 0.014-1.4, e.g. 0.07-0.7, KNU/g (Novo units)). Amylasessuitable are for example Termamyl®, and BAN (Novo Nordisk A/S).Cellulase can for example be used when desired, in an amount in therange of about 0.3 to about 35 CEVU units per gram of the detergentcomposition. Suitable cellulases are for example Celluzyme®, andCarezyme® (Novo Nordisk A/S).

Other enzymes contemplated to be used in the present invention areoxidases and peroxidases

F. Other Optional Components

Other optional components for use in the liquid detergents hereininclude soil removal agents, soil release polymers, antiredepositionagents such as tetraethylene pentamine ethoxylate (from about 0.5% to3%, preferably from about 1% to about 3%, by weight), suds regulants,poly vinyl pyrolidone, carboxy methyl cellulose, clays, and hydrotropessuch as sodium cumene sulfonate, opacifiers, antioxidants, bactericides,dyes, perfumes, and brighteners known in the art. Such optionalcomponents generally represent less than about 15%, preferably fromabout 0.5% to 10%, more preferably from about 1% to about 10%, by weightof the composition.

The compositions may contain from 0% to about 8%, preferably from 0% toabout 5%, by weight of a C₁₂C₁₄ alkenyl succinic acid or salt thereof.These materials are of the general formula R—CH(COOX)CH₂(COOX), whereinR is a C₁₂-C₁₄ alkenyl group and each X is H or a suitable cation, suchas sodium, potassium, ammonium or alkanolammonium (e.g., mono-, di-, ortri-ethanolammonium).

Specific examples are 2-dodecenyl succinate (preferred) and2-tetradecenyl succinate.

The compositions herein optionally contain from about 0.1% to about 1%,preferably from about 0.2% to about 0.6%, by weight of water-solublesalts of ethylenediamine tetramethylenephosphonic acid,diethylenetriamine pentamethylenephosphonic acid, ethylenediaminetetraacetic add (preferred), or diethylenetriamine pentaacetic acid(most preferred) to enhance cleaning performance when pretreatingfabrics.

Furthermore, the detergent compositions may contain from 1-35% of ableaching agent or a bleach precursor or a system comprising bleachingagent and/or precursor with activator therefor.

Further optional ingredients are lather boosters, anti-corrosion agents,soil-suspending agents, sequestering agents, anti-soil redepositionagents, and so on.

The compositions herein preferably contain up to about 10% of ethanol.

G. Other Properties

The instant composition usually has a pH, in a 10% by weight solution inwater at 20° C., of from about 7.0 to 9.0, preferably from about 8.0 toabout 8.5.

The instant compositions can also have a Critical Micelle Concentration(CMC) of less than or equal to 200 parts per million (ppm), and anair/water Interfacial Tension above the CMC of less than or equal to 32,preferably less than or equal to about 30, dynes per centimeter at 35°C. in distilled water. These measurements are described in “Measurementof Interfacial Tension and Surface Tension—General Review for PracticalMan” C. Weser, GIT Fachzeitschrift für das Laboratorium, 24 (1980)642-648 and 734-742, FIT Verlag Ernst Giebeler, Darmstadt, and“Interfacial Phenomena—Equilibrium and Dynamic Effects”, C. A. Millerand P. Neogi, Chapter 1, pp. 29-36 (1985), Marcel Dekker, Inc. New York.

The compositions of the invention can be used for the washing of textilematerials, especially, but without limitation cotton and polyester basedtextiles and mixtures thereof. For example washing processes carried outat temperatures of about 60-65° C. or lower, e.g. about 30-35° C. orlower, are particularly suitable. It can be very suitable to use thecompositions at a rate sufficient to provide about e.g. 0.4-0.8 g/l ofsurfactant in the wash liquor, although it is of course possible to uselower or higher concentrations, if desired. Without limitation it canfor example be stated that a use-rate from about 1 to 10 g/l, e.g. fromabout 3-6 g/l, of the detergent formulation is suitable for use in thecase when the formulations are substantially as in the Examples.

In this aspect the invention is especially related to:

a) A detergent composition formulated as an aqueous detergent liquidcomprising anionic surfactant, nonionic surfactant, humectant, organicacid, caustic alkali, with a pH adjusted to a value between 9 and 10.b) A detergent composition formulated as a non-aqueous detergent liquidcomprising a liquid nonionic surfactant consisting essentially of linearalkoxylated primary alcohol, triacetin, sodium triphosphate, causticalkali, perborate monohydrate bleach precursor, and tertiary aminebleach activator, with a pH adjusted to a value between about 9 and 10.c) An enzymatic liquid detergent composition formulated to give a washliquor pH of 9 or less when used at a rate corresponding to 0.4-0.8 g/lsurfactant.d) An enzymatic liquid detergent composition formulated to give a washliquor pH of 8.5 or more when used at a rate corresponding to 0.4-0.8 μlsurfactant.e) An enzymatic liquid detergent composition formulated to give a washliquor ionic strength of 0.03 or less, e.g. 0.02 or less, when used at arate corresponding to 0.4-0.8 g/l surfactant.f) An enzymatic liquid detergent composition formulated to give a washliquor ionic strength of 0.01 or more, e.g. 0.02 or more, when used at arate corresponding to 0.4-0.8 g/l surfactant.

It was found that the subtilase variants of the present invention canalso be usefully incorporated in detergent composition in the form ofbars, tablets, sticks and the like for direct application to fabrics,hard surfaces or any other surface. In particular, they can beincorporated into soap or soap/synthetic compositions in bar form,wherein they exhibit a remarkable enzyme stability. Detergentcomposition in the form of bars, tablets, sticks and the like for directapplication, are for example described in South African Patent 93/7274,incorporated herein by reference.

Accordingly, the preferred bars in accordance with this inventioncomprise, in addition to the subtilase variant:

i) 25 to 80%, most preferably 25 to 70%, by weight of detergent activewhich is soap or a mixture of soap and synthetic detergent active,reckoned as anhydrous;

ii) 0 to 50% and, most preferably, 10 to 30% by weight of water;

iii) 0 to 35% and, most preferably, 0.1 to 30% by weight filler.

In general, the amount of subtilase variant to be included in suchcompositions of the invention is such that it corresponds with aproteolytic activity of 0.1 to 100 GU/mg based on the composition,preferably 0.5 to 20GU/mg, most preferably 1.0 to 10 GU/mg, where GU/mgis glycine unit per milligram.

Method for Producing Mutations in Subtilase Genes

Many methods for introducing mutations into genes are well known in theart. After a brief discussion of cloning subtilase genes, methods forgenerating mutations in both random sites, and specific sites, withinthe subtilase gene will be discussed.

Cloning Subtilase Genes

The gene encoding a subtilase may be cloned from any of the organismsindicated in Table I, especially gram-positive bacteria or fungus, byvarious methods, well known in the art. First a genomic, and/or cDNAlibrary of DNA must be constructed using chromosomal DNA or messengerRNA from the organism that produces the subtilase to be studied. Then,if the amino-acid sequence of the subtilase is known, homologous,labelled oligonucleotide probes may be synthesized and used to identifysubtilisin-encoding clones from a genomic library of bacterial DNA, orfrom a cDNA library. Alternatively, a labelled oligonucleotide probecontaining sequences homologous to subtilase from another strain ofbacteria or organism could be used as a probe to identifysubtilase-encoding clones, using hybridization and washing conditions oflower stringency.

Yet another method for identifying subtilase-producing clones wouldinvolve inserting fragments of genomic DNA into an expression vector,such as a plasmid, transforming protease-negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for subtilase, such as skim milk. Thosebacteria containing subtilase-bearing plasmid will produce coloniessurrounded by a halo of clear agar, due to digestion of the skim milk byexcreted subtilase.

Generation of Random Mutations in the Subtilase Gene

Once the subtilase gene has been cloned into a suitable vector, such asa plasmid, several methods can be used to introduce random mutationsinto the gene.

One method would be to incorporate the cloned subtilase gene, as part ofa retrievable vector, into a mutator strain of Escherichia coli.

Another method would involve generating a single stranded form of thesubtilase gene, and then annealing the fragment of DNA containing thesubtilase gene with another DNA fragment such that a portion of thesubtilase gene remained single stranded. This discrete, single strandedregion could then be exposed to any of a number of mutagenizing agents,including, but not limited to, sodium bisulfite, hydroxylamine, nitrousacid, formic acid, or hydralazine. A specific example of this method forgenerating random mutations is described by Shortle and Nathans (1978,Proc. Natl. Acad. Sci. U.S.A., 75, 2170-2174). According to the Shortleand Nathans method, the plasmid bearing the subtilase gene would benicked by a restriction enzyme that cleaves within the gene. This nickwould be widened into a gap using the exonuclease action of DNApolymerase I. The resulting single-stranded gap could then bemutagenized using any one of the above mentioned mutagenizing agents.

Alternatively, the subtilisin gene from a Bacillus species including thenatural promoter and other control sequences could be cloned into aplasmid vector containing replicons for both E. coli and B. subtilis, aselectable phenotypic marker and the M13 origin of replication forproduction of single-stranded plasmid DNA upon superinfection withhelper phage IR1. Single-stranded plasmid DNA containing the clonedsubtilisin gene is isolated and annealed with a DNA fragment containingvector sequences but not the coding region of subtilisin, resulting in agapped duplex molecule. Mutations are introduced into the subtilisingene either with sodium bisulfite, nitrous acid or formic acid or byreplication in a mutator strain of E. coli as described above. Sincesodium bisulfite reacts exclusively with cytosine in a single-strandedDNA, the mutations created with this mutagen are restricted only to thecoding regions. Reaction time and bisulfite concentration are varied indifferent experiments such that from one to five mutations are createdper subtilisin gene on average. Incubation of 10 micrograms of gappedduplex DNA in 4 M Na-bisulfite, pH. 6.0, for 9 minutes at 37° C. in areaction volume of 400 microliters, deaminates about 1% of cytosines inthe single-stranded region. The coding region of mature subtilisincontains about 200 cytosines, depending on the DNA strand.Advantageously, the reaction time is varied from about 4 minutes (toproduce a mutation frequency of about one in 200) to about 20 minutes(about 5 in 200).

After mutagenesis the gapped molecules are treated in vitro with DNApolymerase I (Klenow fragment) to make fully double-stranded moleculesand fix the mutations. Competent E. coli are then transformed with themutagenized DNA to produce an amplified library of mutant subtilisins.Amplified mutant libraries can also be made by growing the plasmid DNAin a Mut D strain of E. coli which increases the range of mutations dueto its error prone DNA polymerase.

The mutagens nitrous acid and formic acid may also be used to producemutant libraries. Because these chemicals are not as specific forsingle-stranded DNA as sodium bisulfite, the mutagenesis reactions areperformed according to the following procedure. The coding portion ofthe subtilisin gene is cloned in M13 phage by standard methods andsingle stranded phage DNA prepared. The single-stranded DNA is thenreacted with 1 M nitrous acid pH. 4.3 for 15-60 minutes at 23° C. or 2.4M formic acid for 1-5 minutes at 23° C. These ranges of reaction timesproduce a mutation frequency of from 1 in 1000 to 5 in 1000. Aftermutagenesis, a universal primer is annealed to the M13 DNA and duplexDNA is synthesized using the mutagenized single-stranded DNA as atemplate so that the coding portion of the subtilisin gene becomes fullydouble-stranded. At this point the coding region can be cut out of theM13 vector with restriction enzymes and ligated into an un-mutagenizedexpression vector so that mutations occur only in the restrictionfragment. (Myers et al., Science 229, 242-257 (1985)).

Generation of Site Directed Mutations in the Subtilase Gene

Once the subtilase gene has been cloned, and desirable sites formutation identified and the residue to substitute for the original oneshave been decided, these mutations can be introduced using syntheticoligonucleotides. These oligonucleotides contain nucleotide sequencesflanking the desired mutation sites; mutant nucleotides are insertedduring oligonucleotide synthesis. In a preferred method, a singlestranded gap of DNA, bridging the subtilase gene, is created in a vectorbearing the subtilase gene. Then the synthetic nucleotide, bearing thedesired mutation, is annealed to a homologous portion of thesingle-stranded DNA. The remaining gap is then filled in by DNApolymerase I (Klenow fragment) and the construct is ligated using T4ligase. A specific example of this method is described in Morinaga etal., (1984, Biotechnology 2, 646-639). According to Morinaga et al., afragment within the gene is removed using restriction endonuclease. Thevector/gene, now containing a gap, is then denatured and hybridized to avector/gene which, instead of containing a gap, has been cleaved withanother restriction endonuclease at a site outside the area involved inthe gap. A single-stranded region of the gene is then available forhybridization with mutated oligonucleotides, the remaining gap is filledin by the Klenow fragment of DNA polymerase 1, the insertions areligated with T4 DNA ligase, and, after one cycle of replication, adouble-stranded plasmid bearing the desired mutation is produced. TheMorinaga method obviates the additional manipulation of constructing newrestriction sites, and therefore facilitates the generation of mutationsat multiple sites. U.S. Reissue Pat. No. 34,606 by Estell et al., issuedMay 10, 1994, is able to introduce oligonucleotides bearing multiplemutations by performing minor alterations of the cassette, however, aneven greater variety of mutations can be introduced at any one time bythe Morinaga method, because a multitude of oligonucleotides, of variouslengths, can be introduced.

Expression of Subtilase Mutants

According to the invention, a mutated subtilase gene produced by methodsdescribed above, or any alternative methods known in the art, can beexpressed, in enzyme form, using an expression vector. An expressionvector generally falls under the definition of a cloning vector, sincean expression vector usually includes the components of a typicalcloning vector, namely, an element that permits autonomous replicationof the vector in a microorganism independent of the genome of themicroorganism, and one or more phenotypic markers for selectionpurposes. An expression vector includes control sequences encoding apromoter, operator, ribosome binding site, translation initiationsignal, and, optionally, a repressor gene or various activator genes. Topermit the secretion of the expressed protein, nucleotides encoding a“signal sequence” may be inserted prior to the coding sequence of thegene. For expression under the direction of control sequences, a targetgene to be treated according to the invention is operably linked to thecontrol sequences in the proper reading frame. Promoter sequences thatcan be incorporated into plasmid vectors, and which can support thetranscription of the mutant subtilase gene, include but are not limitedto the prokaryotic beta-lactamase promoter (Villa-Kamaroff, et al.(1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3727-3731) and the tac promoter(DeBoer, et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 21-25). Furtherreferences can also be found in “Useful proteins from recombinantbacteria” in Scientific American (1980) 242, 74-94.

According to one embodiment B. subtilis is transformed by an expressionvector carrying the mutated DNA. If expression is to take place in asecreting microorganism such as B. subtilis a signal sequence may followthe translation initiation signal and precede the DNA sequence ofinterest. The signal sequence acts to transport the expression productto the cell wall where it is cleaved from the product upon secretion.The term “control sequences” as defined above is intended to include asignal sequence, when it is present.

Other host systems known to the skilled person are also contemplated forthe expression and production of the protease variants of the invention.Such host systems comprise fungi, including filamentous fungi, plant,avian and mammalian cells, as well as others.

Materials and Methods Strains:

B. subtilis 309 and 147 are variants of Bacillus lentus, deposited withthe NCIB and accorded the accession numbers NCIB 10309 and 10147, anddescribed in U.S. Pat. No. 3,723,250 incorporated by reference herein.

E. coli MC 1000 (M. J. Casadaban and S. N. Cohen (1980); J. Mol. Biol.138 179-207), was made r⁻,m⁺ by conventional methods and is alsodescribed in U.S. Patent Application Ser. No. 039,298.

Proteolytic Activity

In the context of this invention proteolytic activity is expressed inKilo NOVO Protease Units (KNPU). The activity is determined relativelyto an enzyme standard (SAVINASE™), and the determination is based on thedigestion of a dimethyl casein (DMC) solution by the proteolytic enzymeat standard conditions, i.e. 50° C., pH 8.3, 9 min. reaction time, 3min. measuring time. A folder AF 220/1 is available upon request to NovoNordisk A/S, Denmark, which folder is hereby included by reference.

A GU is a Glycine Unit, defined as the proteolytic enzyme activitywhich, under standard conditions, during a 15 minutes' incubation at 40°C., with N-acetyl casein as substrate, produces an amount of NH₂-groupequivalent to 1 μmole of glycine.

Enzyme activity can also be measured using the PNA assay, according toreaction with the soluble substratesuccinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol, whichis described in the Journal of American Oil Chemists Society, Rothgeb,T. M., Goodlander, B. D., Garrison, P. H., and Smith, L. A., (1988).

EXAMPLES

For the generation of enzyme variants according to the invention thesame materials and methods as described in i.a. WO 89/06279 (NovoNordisk A/S), EP 130,756 (Genentech), EP 479,870 (Novo Nordisk A/S), EP214,435 (Henkel), WO 87/04461 (Amgen), WO 87/05050 (Genex), EPapplication no. 87303761 (Genentech), EP 260,105 (Genencor), WO 88/06624(Gist-Brocades NV), WO 88/07578 (Genentech), WO 88/08028 (Genex), WO88/08033 (Amgen), WO 88/08164 (Genex), Thomas et al. (1985) Nature, 318375-376; Thomas et al. (1987) J. Mol. Biol., 193, 803-813; Russel andFersht (1987) Nature 328 496-500. Other methods well established in theart may also be used.

Example 1 Construction and Expression of Enzyme Variants

A vector suited to a synthetic gene coding for subtilase 309 and itsmutants was constructed. It is essentially a pUC19 plasmid[Yanish-Perron and Messing (1985) Gene; 33 103-119], in which themultiple cloning site has been replaced by a linker containing therestriction sites used to separate five sub-fragments constituting thegene. The new linker was inserted into EcoRI-HindIII cut pUC19 therebydestroying these sites. The details of this construction are describedin WO 92/19729 on pages 25-26 and in FIG. 1 (sheets 1/7-7/7) thereof,the content of which is hereby included by reference.

Each subfragment was made from 6 to 12 oligonucleotides. Theoligonucleotides were synthesized on an automatic DNA synthesizer usingphosphoramidite chemistry on a controlled glass support [Beaucage andCarruthers (1981); Tetrahedron Letters 22 1859-1869].

The five subfragments were isolated on a 2% agarose gel and insertedinto pSX191. The sequence was verified by dideoxynucleotide sequencing.Fragments A-E were isolated and ligated together with KpnI-BamHI cutpSX191. The ligation mixtures were used to transform competent E. coliMC1000 r⁻,m⁺ selecting for ampicillin resistance. The 850 bp KpnI-BamHIfragment that constitutes the part of the subtilisin 309 gene coding forthe mature part of the enzyme was then used to replace the wild typegene on pSX212 giving rise to pSX222, which was then transformed into acompetent B. subtilis strain. After fermentation of the transformedstrain and purification of the enzyme it was shown that the product wasindistinguishable from the wild type product.

Protease variants derived from the synthetic gene are made by usingoligonucleotides with altered sequence at the place(s) where mutation iswanted (e.g. with sequences as given below) and mixing them with therest of the oligonucleotides appropriate to the synthetic gene. Assemblyof the variant gene is carried out with the variant materials in amanner otherwise analogous to that described above. Further informationon synthetic genes generally is available in Agarval et al. (1970);Nature; 227, 27-34.

A KpnI site was introduced into the beginning of the subtilase 309synthetic gene encoding the mature part of the enzyme. The method usedis called oligonucleotide directed double-strand break repairmutagenesis and is described by Mandecki (1986) Proc. Nat. Acad. Sci.USA 83 7177-7181. pSX172 is opened with NcoI at the beginning of themature part of the subtilase 309 gene and is mixed with theoligonucleotide NOR 789 (see WO 92/19729), heated to 100° C., cooled to0° C., and transformed into E. coli. After retransformation, therecombinants can be screened by colony hybridisation using 32-P-labelledNOR 789. The recombinants that turned out to be positive during thescreening had the KpnI site introduced right in front of NcoI bychanging two bases without changing the amino acid sequence. pSX172 isdescribed in EP 405 901. The KpnI site so created is inserted intopSX120 on a 400-bp PvuI-NheI fragment, giving rise to pSX212. pSX120 isalso described in EP 405 901.

The synthetic gene is inserted between KpnI and BamHI on pSX212, givingrise to pSX222.

Examples of mutations and corresponding sequences of oligonucleotidesare-as follows:

These oligonucleoties were combined with the rest of theoligonucleotides from the synthetic gene that was not changed.

Example 2 Purification of Enzyme Variants

This procedure relates to purification of a 10 liter scale fermentationof subtilisin 147, subtilisin 309 or mutants thereof.

Approximately 8 liters of fermentation broth were centrifuged at 5000rpm for 35 minutes in 1 liter beakers. The supernatants were adjusted topH 6.5 using 10% acetic acid and filtered on Seitz Supra S100 filterplates.

The filtrates were concentrated to approximately 400 ml using an AmiconCH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UFconcentrate was centrifuged and filtered prior to absorption at roomtemperature on a Bacitracin affinity column at pH 7. The protease waseluted from the Bacitracin column at room temperature using 25%2-propanol and 1 M sodium chloride in a buffer solution with 0.01dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chlorideadjusted to pH 7.

The fractions with protease activity from the Bacitracin purificationstep were combined and applied to a 750 ml Sephadex G25 column (5 cmdia.) equilibrated with a buffer containing 0.01 dimethylglutaric acid,0.2 M boric acid and 0.002 M calcium chloride adjusted to pH 6.5.

Fractions with proteolytic activity from the Sephadex G25 column werecombined and applied to a 150 ml CM Sepharose CL 6B cation exchangecolumn (5 cm dia.) equilibrated with a buffer containing 0.01 Mdimethylglutaric acid, 0.2 M boric acid, and 0.002 M calcium chlorideadjusted to pH 6.5.

The protease was eluted using a linear gradient of 0-0.1 M sodiumchloride in 2 liters of the same buffer (0-0.2 M sodium chloride in caseof subtilisin 147).

In a final purification step protease containing fractions from the CMSepharose column were combined and concentrated in an Amiconultrafiltration cell equipped with a GR81PP membrane (from the DanishSugar Factories Inc.).

By using the techniques of Example 1 for the construction and the aboveisolation procedure the following subtilisin 309 variants were producedand isolated:

A: G159I B: S164I C: Y167I D: R170I E: R170L F: R170M G: R170F H: G195FI: S57P+R170L J: R170L+N218S K: S57P+R170L+N218S L: R170L+N218S+M222A M:S57P+R170L+S188P+A194P N: Y167I+R170L O: S57P+R170L+Q206E P: R170L+Q206EQ: Y167I+R170L+Q206E R: Y167I+R170L+A194P S: Y167I+R170L+N218S T:Y167I+R170L+A194P+N218S U: Y167I+Y171I V: R170G W: R170C X: Y171I Y:Y167I+R170L+N218S Example 3 Stability in Detergent CompositionsComprising Enzyme Variants Example D1

An (isotropic) aqueous detergent liquid according to an embodiment ofthe invention is formulated to contain:

Ingredient % NaLAS 8.0 Neodol 25-9 8.0 AES 25-3S 14.0 NaCitrate•2H₂O 5.0Propylene Glycol 5.0 Sorbitol 4.5 F-dye Tinopal UNPA-GX 0.15 Lytron 614Opacifier 0.03 Kathon Preservative 0.0003 Acid Blue 80 0.00117 AcidViolet 48 0.0033 SAVINASE ® 16L 0.25 LIPOLASE ® 100L 0.70 Fragrance 0.15Water ad 100.0

The pH is adjusted to 7.1.

TABLE III Residual enzyme activity (in percentage or original activity)after storage at 37° C. for Example D1 comprising the BLS309 variantS57P + R170L + N218S. Storage time (days) Wild-type S57P + R170L + N218S0 100 100 3 44 74 7 11 50 10 5 36 14 7 27

From Table III it is evident that the variant S57P+R170L+N218S exhibitsa remarkably improved stability in this type of detergent. Moreover, thevariant S57P+R170L+N218S possesses excellent compatibility towardslipase.

TABLE IV Residual lipase activity (in percentage of original activity)after storage at 37° C. for Example D1 comprising the BLS309 variantS57P + R170L + N218S and LIPOLASE ®. Storage time (days) LIPOLASE ®plus: Wild-type S57P + R170L + N218S 0 100 100 3 38 67 7 24 44 10 22 3314 21 27

From Table IV it is apparent that, in addition to the stability of theprotease, the compatibility of the protease is also improved.

Example D2

A non-aqueous detergent liquid according to an embodiment of theinvention is formulated using 38.5% C13-C15 linear primary alcoholalkoxylated with 4.9 mol/mol ethylene oxide and 2.7 mol/mol propyleneoxide, 5% triacetin, 30% sodium triphosphate, 4% soda ash, 15.5% sodiumperborate monohydrate containing a minor proportion of oxoborate, 4%TAED, 0.25% EDTA of which 0.1% as phosphonic acid, Aerosil 0.6%, SCMC1%, and 0.6% protease. The pH is adjusted to a value between 9 and 10,e.g. about 9.8.

Example D3

Structured liquid detergents can for example contain, in addition to aprotease as described herein, 2-15% nonionic surfactant, 5-40% totalsurfactant, comprising nonionic and optionally anionic surfactant, 5-35%phosphate-containing or non-phosphate containing builder, 0.2-0.8%polymeric thickener, e.g. cross-linked acrylic polymer with m.w. over10⁶, at least 10% sodium silicate, e.g. as neutral waterglass, alkali(e.g. potassium-containing alkali) to adjust to desired pH, preferablyin the range 9-10 or upwards, e.g. above pH 11, with a ratio sodiumcation:silicate anion (as free silica) (by weight) less than 0.7:1, andviscosity of 0.3-30 Pas (at 20° C. and 20 s-⁻¹).

Suitable examples contain about 5% nonionic surfactant C13-15 alcoholalkoxylated with about 5 EO groups per mole and with about 2.7 PO groupsper mole, 15-23% neutral waterglass with 3.5 weight ratio between silicaand sodium oxide, 13-19% KOH, 8-23% STPP, 0-11% sodium carbonate, 0.5%Carbopol 941 (TM).

Protease may be incorporated at for example 0.5%.

Example D4

(Decoupling polymer liquid) Priolene 6907 4.5 KOH 10 EthoxylatedAlcohol•7EO (Synperonic A7) 4.5 Ethoxylated Alcohol•3EO (Synperonic A3)4.5 Zeolite 4A 15 Fluorescer Tinopal CBS-X 0.08 Narlex DC1 1 Citric acid8.23 Antifoam silicone DB100 0.3 LAS acid 16.5 Perfume 0.5 Water to 100

TABLE V Residual enzyme activity (in percentage of original activity)after storage at 37° C. for Example D4 comprising the R170L variant ofBLS309. Storage time (days) R170L Wild-type 0 100 100 2 98 73 4 96 66 1094 46 33 87 8 81 78 2.1 101 71 0

From Table V it is evident that the R170L variant exhibits a remarkablyimproved stability in this type of detergent.

TABLE VI Enzyme Storage Y167I + R170L + (days) WT R170M S57P + R170L +Q206E N218S 0 100 100 100 100 0.1 90.2 78 97 94 1 58 53 95 68 2 40 34 8755 5 16 27 75 29 6 12 22 73 24 8 8 19 77 17 14 2 11 52 4

From Table VI it can be seen that the variants tested exhibit improvedstability in comparison to the wild type enzyme in this type ofdetergent

Example D5

(Decoupling polymer liquid) Priolene 6907 4.5 KOH 10 EthoxylatedAlcohol•7EO (Synperonic A7) 4.5 Ethoxylated Alcohol•3EO (Synperonic A3)4.5 Zeolite 4A 15 Fluorescer Tinopal CBS-X 0.08 Narlex DC1 1 Citric acid8.23 Antifoam silicone DB100 0.3 LAS acid 16.5 Lipolase ® 100L 0.6Perfume 0.5 Water to 100

TABLE VII Residual enzyme activity (in percentage of original activity)after storage at 37° C. for Example D5 comprising the BLS309 variantS57P + R170L + N218S. Residual protease Residual lipase Storage activityactivity time S57P + R170L + S57P + R170L + (days) N218S Wild-type R170LN218S 0 100 100 100 100 2 — 27 41 94 5 97 9 15 76 8 87 4 7 71 12 91 2.412 78 28 100 2.4 12 70

From Table VII it is evident that the variant S57P+R170L+N218S exhibitsa remarkably improved stability in this type of detergent. Moreover thevariant S57P+R170L+N218S possesses excellent compatibility towardslipase.

Example D6

Soap bars were produced containing 49.7 wt. 80/20 tallow/coconut soap,49.0% water, 20% sodium citrate, 1.0% citric acid and 0.031% protease.After preparation of the soap bars they were stored at ambienttemperature and after specific time intervals samples were taken andmeasured for protease activity. The stability data are given below:

TABLE VIII Enzyme Storage R170L + N218S + (days) WT R170L S57P R170L +Y167I 0 100 100 100 100 1 50 100 97 94 2 25 91 100 83 3 — 100 94 80 6 —98 89 90 10 0 100 94 71 17 — 93 80 73 27 — 95 86 70

From Table VIII it is evident that the subtilase variants R170L,R170L+N218S+S57P and R170L+Y167I exhibit a remarkably improved stabilityin this type of detergent.

Example D7

Soap bars were produced containing 63.88% 80/20 tallow/coconut soap, 1%coconut fatty acid, 25.1% water, 10% sodium citrate and 0.021% protease.The laundry soap bars were stored at 37° C. and after specific timeintervals samples were taken and measured for protease activity.

TABLE IX Stability data: Enzyme Storage (days) WT R170L + N218S + S57P 0100 100 10 10 90.1 14 — 81.5 20 0 91.4 31 — 72.8 35 — 79 45 — 78

From Table IX it is evident that the subtilase variant R170L+N218S+S57Pexhibits a remarkably improved stability in this type of detergent.

Example 4 Wash Performance of Detergent Compositions Comprising EnzymeVariants

The following examples provide results from a number of washing teststhat were conducted under the conditions indicated.

Experimental Conditions

TABLE X Experimental conditions for evaluation of Subtilisin 309variants. Detergent Protease model detergent ′95 Detergent dose 3 g/l pH9.5 Wash time 15 min. Temperature 15° C. Water hardness 9° dH ~1.61 mMCa²⁺/Mg²⁺ Enzymes Subtilisin 309 variants as listed below Enzyme conc.0; 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 3.0 mg/l Test system 150 mlbeakers with a stirring rod. Cloth/volume 5 cloths (Ø 2.5 cm)/50 mlDetergent solution. Cloth Cotton soiled with grass juice

Subsequent to washing the cloths were flushed in tap water andair-dried.

The above model detergent is a simple detergent formulation. The mostcharacteristic features are that STP is used as builder and the contentof anionic tenside (LAS) is quite high. Further the pH is adjusted to9.5, which is low for a powder detergent.

Table XI

The composition of the model detergent is as follows:

25% STP (Na₅P₃O₁₀) 25% Na₂SO₄ 10% Na₂CO₃ 20% LAS (Nansa 80S) 5% NI(Dobanol 25-7) 5% Na₂Si₂O₅ 0.5% Carboxymethylcellulose (CMC)

9.5% waterdose: 3 g/lpH is adjusted to 9.5

Measurement of remission (R) on the test material has been done at 460nm using an Elrepho 2000 photometer (without UV). The measured valueshave been fitted to the expression:

ΔR=(a·ΔR _(max) ·c)/(ΔR _(max) +a·c)

The improvement factor is calculated by use of the initial slope of thecurve: IF=a/a_(ref).

ΔR is the wash effect of the enzyme in remission units.a is the initial slope of the fitted curve (c→0).a_(ref.) is the initial slope for the reference enzyme.c is the enzyme concentration in mg/lΔR_(max) is the theoretical maximum wash effect of the enzyme inremission units (c→∞).

TABLE XII Variants and improvement factors for subtilisin 309.Designation Variant IF S003* R170Y 2.8 S004* R170Y + G195E 2.6 S012*R170Y + G195E + K251E 1.6 G R170F 3.3 E R170L 3.8 F R170M 2.4 D R170I4.1 I S57P + R170L 3.9 J R170L + N218S 1.6 K S57P + R170L + N218S 2.3 NY167I + R170L 6.2 P R170L + Q206E 2.6 V R170G 2.0 W R170C 3.4 O S57P +R170L + Q206E 2.9 Q Y167I + R170L + Q206E 2.4 R Y167I + R170L + A194P5.1 X Y171I 1.2 Y Y167I + R170L + N218S 4.0 T Y167I + R170I + A194P +N218S 3.6 *Described in WO 91/00345

As it can be seen from Table XII all the subtilisin 309 variants of theinvention exhibits an improvement in wash performance.

TABLE XIII Variants and improvement factors for subtilisin 309 in adetergent as described in Example D4 Designation Variant IF S003* R170Y1.5 F R170M 1.2 O S57P + R170L + Q206E 5.0 X Y171I 4.2 R Y167I + R170L +A194P 1.2 T Y167I + R170L + A194P + N218S 2.0 Y Y167I + R170L + N218S2.3 *Described in WO 91/00345

As it can be seen from Table XIII all the Subtilisin 309 variants of theinvention exhibits an improvement in wash performance.

1-87. (canceled)
 88. A modified subtilase comprising one or more of thefollowing substitutions: (a) a substitution of the amino acid residue atposition 167 with Met, Pro, or Trp, (b) a substitution of the amino acidresidue at position 170 with Ile, Met, or Val, (c) a substitution of theamino acid residue at position 171 with Met, Pro or Trp, and (d) asubstitution of the amino acid residue at position 194 with Ile, Met,Phe, Trp, or Val, wherein each position corresponds to a position of theamino acid sequence of subtilisin BPN′ (SEQ ID NO: 7).
 89. The modifiedsubtilase of claim 88, which comprises a substitution of the amino acidresidue at position 167 with Met, Pro, or Trp.
 90. The modifiedsubtilase of claim 88, which comprises a substitution of the amino acidresidue at position 170 with Ile, Met, or Val.
 91. The modifiedsubtilase of claim 88, which comprises a substitution of the amino acidresidue at position 171 with Met, Pro or Trp.
 92. The modified subtilaseof claim 88, which comprises a substitution of the amino acid residue atposition 194 with Ile, Met, Phe, Trp, or Val.
 93. The modified subtilaseof claim 88, further comprising at least one further mutation at one ormore of positions: 27, 36, 57, 76, 97, 101, 104, 120, 123, 206, 218,222, 224, 235 and
 274. 94. The modified subtilase of claim 93, whereinat least one further mutation is selected from the group consisting ofK27R, *36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S,Q206E, N218S, M222A, M222S, T224S, K235L, and T274A.
 95. A detergentcomposition comprising a modified subtilase of claim 88 and asurfactant.
 96. A modified subtilisin 309 comprising one or both of thefollowing substitutions: (a) a substitution of Glu at position 136 withAla, Asn, Cys, Gly, His, Ser, Thr, or Tyr, and (b) a substitution atY171A, Y171C, Y171G, Y171H, Y171I, Y171L, Y171M, Y171N, Y171P, Y171Q,Y171S, or Y171W, wherein each position corresponds to a position of theamino acid sequence of subtilisin BPN′ (SEQ ID NO: 7).
 97. The modifiedsubtilisin 309 of claim 96, which comprises a substitution of Glu atposition 136 with Ala, Asn, Cys, Gly, His, Ser, Thr, or Tyr.
 98. Themodified subtilisin 309 of claim 96, which comprises Y171A, Y171C,Y171G, Y171H, Y171I, Y171L, Y171M, Y171N, Y171P, Y171Q, Y171S, or Y171W.99. The modified subtilisin 309 of claim 96, further comprising at leastone further mutation at one or more of positions: 27, 36, 57, 76, 97,101, 104, 120, 123, 194, 206, 218, 222, 224, 235 and
 274. 100. Themodified subtilisin 309 of claim 99, wherein at least one furthermutation is selected from the group consisting of K27R, *36D, S57P,N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, A194P, Q206E,N218S, M222A, M222S, T224S, K235L, and T274A.
 101. A detergentcomposition comprising a modified subtilisin 309 of claim 96 and asurfactant.
 102. A modified subtilisin 147 comprising one or both of thefollowing substitutions: (a) a substitution of Glu at position 136 withAla, Asn, Cys, Gly, His, Ser, Thr, or Tyr, (b) a substitution at Y171A,Y171C, Y171G, Y171H, Y171I, Y171L, Y171M, Y171N, Y171P, Y171Q, Y171S, orY171W, wherein each position corresponds to a position of the amino acidsequence of subtilisin BPN′ (SEQ ID NO: 7).
 103. The modified subtilisin147 of claim 102, further comprising at least one further mutation atone or more of positions: 27, 36, 57, 76, 97, 101, 104, 120, 123, 194,206, 218, 222, 224, 235 and
 274. 104. The modified subtilisin 147 ofclaim 103, wherein at least one further mutation is selected from thegroup consisting of K27R, *36D, S57P, N76D, G97N, S101G, V104A, V104N,V104Y, H120D, N123S, A194P, Q206E, N218S, M222A, M222S, T224S, K235L,and T274A.
 105. A detergent composition comprising a modified subtilisin147 of claim 102 and a surfactant.
 106. A modified Bacillus proteasePB92 comprising a substitution Y171A, Y171C, Y171G, Y171H, Y171I, Y171L,Y171M, Y171N, Y171P, Y171Q, Y171S, or Y171W, wherein each positioncorresponds to a position of the amino acid sequence of subtilisin BPN′(SEQ ID NO: 7).
 107. The modified Bacillus protease PB92 of claim 106,further comprising at least one further mutation at one or more ofpositions: 27, 36, 57, 76, 97, 101, 104, 120, 123, 194, 206, 218, 222,224, 235 and
 274. 108. The modified Bacillus protease PB92 of claim 107,wherein at least one further mutation is selected from the groupconsisting of K27R, *36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y,H120D, N123S, A194P, Q206E, N218S, M222A, M222S, T224S, K235L, andT274A.
 109. A detergent composition comprising a modified Bacillusprotease PB92 of claim 106 and a surfactant.
 110. A modified subtilisinBPN′ comprising a substitution of Lys at position 136 with Ala, Asn,Cys, Gly, His, Ser, Thr, or Tyr, wherein each position corresponds to aposition of the amino acid sequence of subtilisin BPN′ (SEQ ID NO: 7).111. The modified subtilisin BPN′ of claim 110, further comprising atleast one further mutation at one or more of positions: 27, 36, 57, 76,97, 101, 104, 120, 123, 194, 206, 218, 222, 224, 235 and
 274. 112. Themodified subtilisin BPN′ of claim 111, wherein at least one furthermutation is selected from the group consisting of K27R, *36D, S57P,N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, A194P, Q206E,N218S, M222A, M222S, T224S, K235L, and T274A.
 113. A detergentcomposition comprising a modified subtilisin BPN′ of claim 111 and asurfactant.
 114. A modified subtilisin Carlsberg comprising asubstitution of Lys at position 136 with Ala, Asn, Cys, Gly, His, Ser,Thr, or Tyr, wherein the position corresponds to a position of the aminoacid sequence of subtilisin BPN′ (SEQ ID NO: 7).
 115. The modifiedsubtilisin Carlsberg of claim 114, further comprising at least onefurther mutation at one or more of positions: 27, 36, 57, 76, 97, 101,104, 120, 123, 194, 206, 218, 222, 224, 235 and
 274. 116. The modifiedsubtilisin Carlsberg of claim 115, wherein at least one further mutationis selected from the group consisting of K27R, *36D, S57P, N76D, G97N,S101G, V104A, V104N, V104Y, H120D, N123S, A194P, Q206E, N218S, M222A,M222S, T224S, K235L, and T274A.
 117. A detergent composition comprisinga modified subtilisin Carlsberg of claim 114 and a surfactant.